CN103739714B - The fusion rotein of TNF �� and DC-SIGN and application thereof - Google Patents

The present invention relates to the fusion rotein of TNF �� and DC-SIGN and application thereof. Normal cell is easily caused damage by traditional antitumor drug or methods for the treatment of in the process of killing tumor cell. The targeting anti-tumor fusion rotein of the present invention, it is by recognition function territory and action function territory, and connects the connection peptides composition of these two functional domains. Described recognition function territory is the agglutinin peptide with tumor cell surface sugar chain identification and combined function--between the specific cell on DC surface, adhesion factor 3 is in conjunction with nonconformity element molecule (DC-SIGN, dendritic-cell-specific? ICAM-3-grabbing? nonintegrin), it is positioned at the C end of fusion rotein; Described action function territory is tumour necrosis factor (TNF), is positioned at the N end of fusion rotein; Described connection peptides is for containing 8-25 amino acid whose short peptide. This fusion rotein can utilize lectin and cancer cells to carry out target to combination, by the lethal effect reducing normal tissue and cell of short apoptosis part cancer cell specific induction of apoptosis simultaneously.

The fusion rotein of TNF �� and DC-SIGN and application thereof

Technical field

The present invention relates to genetically engineered field, relate to fusion rotein and the application thereof of TNF �� and DC-SIGN, more specifically, the present invention relates to the fusion rotein of extracellular region structural domain in conjunction with nonconformity element molecule (DC-SIGN, dendritic-cell-specificICAM-3-grabbingnonintegrin) of adhesion factor 3 between the specific cell comprising the DC surface identifying variation or high expression level sugar chain, connection peptides and tumour necrosis factor (TNF).

Background technology

Along with people's living environment and living-pattern preservation, antitumor market progressively expands, the conventional meanses such as the radiation and chemotherapy of current treatment tumour cannot distinguish tumour cell and normal tissue cell, usually cause severe side effect, while killing tumor cell, often also destroy the normal immunologic function of body, the existence of patient, quality of life are declined greatly. Therefore the toxic side effect reducing present anti-tumor medicine becomes the problem needing solution badly, and current research has shown that targeting antineoplastic medicine thing has the feature of toxic side effect efficient, low and low cost oncotherapy, has become the focus of recent research.

Difference according to recognition site, targeted anticancer medicine mainly comprises monoclonal antibody and small molecular protein kinase inhibitor. The mechanism of action of monoclonal antibody is that the tumour antigen utilizing tumor cell surface to have some special can be used as attack target spot, and small molecular protein kinase inhibitor energy targeted inhibition abnormal protein kinase acceptor, thus the biological procedureses such as the growth of Tumor suppression, differentiation, metabolism and play antineoplastic curative effect. But, monoclonal antibody medicine and kinase inhibitor drug on tumor cell only have single recognition site, and recognition site has sudden change or the cancer cell fragmentation effect of structural modification limited, mostly are combination therapy medicine on therefore clinical, and its market is many is monopolized by transnational company, price is very expensive. Therefore, exploitation targeting and the more cheap targeting anti-tumor medicine of better efficacy, price will improve the quality of production of malignant tumor patient greatly, reduce the medical expense expense of patient.

Lectin (lectin) is that a class can optionally identify non-enzymatic that is sugared and non-covalent Reversible binding with it, non-antibody protein. DC-SIGN/CD209(DendriticCell-SpecificIntercellularadhesio nmolecule-3-GrabbingNon-integrin) structure in containing the carbohydrate identified region (carbohydraterecognitiondomain of calcium dependent form, CRD), belong to the category of C type lectin. DC-SIGN can broad incorporation pathogenic agent source carbohydrate structure, as included the sugared binding substances of seminose and comprise Louis these blood group antigen (LewbloodgroupAgs) Le of Fucose^x��Le^y��Le^aAnd Le^b(M.Nonakaetal., JBiolChem286,22403). DC-SIGN also has specific recognition and the ability in conjunction with tumour cell sugar mark, and these tumour sugar marks comprise CEA, Lewis blood group antigen, Mac-2BP, Span-1 etc. This kind realizes in combination with CRD region and mutual identification of tumour-specific sugar chain (Man, D-GlcNAc, Fuc) of C type lectin.

Apoptosis (Apoptosis) is the process withered died by the active that the energy dependence cell of Gene Handling is orderly. Apoptotic pathways can be caused after apoptosis factor and receptors bind, effectively suppress or killer cell, also can be used for the research and development of anti-cancer agent. Tumour necrosis factor (TNF) is as the strongest cytokine of the anti-tumor activity found so far, and as far back as the eighties in last century, it is once launched clinical study by America and Europe, but due to its biological effect extensively, toxic side effect is relatively big and is forced to terminate. Therefore, how to reduce under the prerequisite ensureing TNF response to treatment its toxic side effect become tnf family cytokines member can as the critical problem of antitumor drug. Patent CN1458977A and CN1865444A once adopted the strand of monoclonal antibody as target to structural domain and TNF �� amalgamation and expression. The target function of this kind of fusion rotein depends on the targeting of single-chain antibody (scFv). But, single-chain antibody complex structure, binding ability are poor, unstable expression, and recognition site is single, antigen site namely cannot reach as undergone mutation etc. target to effect, thus had influence on the overall function of monoclonal antibody. Therefore, develop a kind of have tumour cell high specific recognition, stable antitumor drug there is important meaning.

Summary of the invention

For overcoming above-mentioned deficiency, the present invention's first object is to provide a kind of targeting anti-tumor fusion rotein, and it is by recognition function territory and action function territory, and connects the connection peptides composition of these two functional domains. Described recognition function territory is C type lectin, is positioned at the C end of fusion rotein; Described action function territory is tumour necrosis factor, is positioned at the N end of fusion rotein; Described connection peptides is for containing 8-25 amino acid whose short peptide.

Preferably, recognition function territory described above is DC-SIGN/CD209. Preferred, recognition function territory described above is the extracellular region of DC-SIGN/CD209.

Preferably, action function territory described above is TNF ��. Preferred, action function territory described above is the extracellular region of TNF ��.

Preferably, connection peptides described above is flexible peptide linker.

Preferably, the aminoacid sequence of connection peptides described above is GGGGGGGGGG or (EAAAK)_nOr (GGGGS)_n, wherein n=3,4 or 5.

Preferably, the aminoacid sequence of fusion rotein described above is as shown in SEQIDNO:2 or SEQIDNO:4.

It is a further object to provide a kind of gene expressing targeting anti-tumor fusion rotein described above, described gene is preferably as shown in SEQIDNO:1 or SEQIDNO:3.

It is a further object to provide a kind of plasmid containing gene described above. Preferably, described plasmid is p3FLAG-CMV-13 or pCHO1.0.

It is a further object to provide a kind of mammalian cell containing plasmid described above.

It is a further object of the present invention to provide a kind of pharmaceutical preparation or the pharmaceutical composition that comprise fusion rotein described above. Described pharmaceutical composition can also comprise at least one other treatment agent, such as antibody, kinase inhibitor or cancer vaccine etc.

It is a further object of the present invention to provide fusion rotein described above at the novelty teabag preparing in antitumor drug. Preferably, described novelty teabag is treatment large bowel cancer.

Tumour necrosis factor is connected by connection peptides by the present invention with C type lectin. This kind of fusion rotein utilizes the target of C type lectin and cancer cells to the apoptosis effect cancer cell specific induction of apoptosis combined and tumour necrosis factor mediates simultaneously. The combination of target function and short apoptosis capacity makes such fusion rotein while specific killing cancerous tumor cell, reduces the popularity of its effect as much as possible, it is ensured that healthy tissues and cell are not killed. In addition, contriver is also unexpected finds the character that the fusion of C type lectin also imparts albumen multimerization. The fusion rotein forming polymer adds the short apoptotic signal of local, thus strengthen the cross reaction of tumour cell and immunocyte, facilitate the anti-power of antineoplastic immune of body to a great extent so that the biological activity of fusion rotein entirety is greatly improved.

Accompanying drawing explanation

Fig. 1 is multisequencing comparison chart. FP: fusion rotein; TNF ��: tumour necrosis factor; Linker:GGGGGGGGGG connection peptides (is called for short G10); Between the specific cell on DC-SIGN:DC surface, adhesion factor 3 is in conjunction with nonconformity element molecule.

Fig. 2 is the structural simulation figure of TNF ��-G10-DC-SIGN fusion rotein. G1-L177:TNF ��; G178-G187:G10 connection peptides; Q188-A533:DC-SIGN.

Fig. 3 is the TNF �� structural simulation comparison in TNF �� monomer and fusion rotein TNF ��-G10-DC-SIGN. Selecting 33-177 amino acid to carry out structure alignment, gained RMSD value isShow that functional domain and the monomeric protein TNF �� of the TNF �� in fusion rotein do not have anything to change. As can be seen from the figure TNF �� almost overlaps completely with the TNF �� end of AT3002 fusion rotein. Therefore, it is possible to speculating that the albumen tertiary structure of the TNF �� end in AT3002 fusion rotein and TNF �� monomer is basically identical, namely the TNF �� in AT3002 fusion rotein still remains the native biological activity of TNF ��. Illustrated blocks region representation TNF �� acceptor and TNF �� interaction region.

Fig. 4 is that fusion rotein docks detail view with acceptor, is the partial enlargement figure of the boxed area of Fig. 3. Wherein upper part is fusion rotein AT3002, and lower part is TNF �� acceptor. TNF �� in visible fusion rotein AT3002 and the combination between its acceptor are without space steric hindrance, it is possible to interact.

Fig. 5 represents AT3002 fusion rotein codon optimized front and back nucleotide sequence comparison.

Wherein, odd number capable (row that namely " original series " is corresponding) is the sequence before the optimization of AT3002 gene codon; Even number line (row that namely " majorizing sequence " is corresponding) is the sequence after the optimization of AT3002 gene codon.

Fig. 6 AT3002 gene codon optimizes front and back CAI index in mammalian cell expression host.

Wherein, Fig. 6-a represent AT3002 gene nucleotide series in mammalian cell expression host CAI index through program computation be 0.84; Fig. 6-b represent the AT3002 gene nucleotide series codon of the present invention after optimization in mammalian cell expression host CAI index through program computation be 0.85.

Fig. 7 is that AT3002 gene codon optimizes front and back optimal codon frequency distribution areal map in mammalian cell expression host.

Wherein, Fig. 7-a represents AT3002 gene nucleotide series optimal codon frequency distribution areal map in mammalian cell expression host, as can be seen from the figure: the low utilization ratio codon of AT3002 gene nucleotide series occurs that per-cent is 5%; Fig. 7-b represents the AT3002 gene of the present invention after optimization optimal codon frequency distribution areal map in mammalian cell expression host, and the low utilization ratio codon of the AT3002 gene-code subsequence of the present invention after optimization occurs that per-cent is 1%.

Fig. 8 is that AT3002 gene codon optimizes front and back average GC base contents distributed areas figure in mammalian cell expression host.

Wherein, Fig. 8-a represents that AT3002 gene nucleotide series average GC base contents in mammalian cell expression host is: 57.25%; Fig. 8-b represents that the AT3002 gene codon of the present invention after optimization average GC base contents in mammalian cell expression host is: 54.57%.

Fig. 9 is carrier for expression of eukaryon structural representation.

Wherein, Fig. 9-a is the structural representation of carrier for expression of eukaryon p3xFLAG-CMV-13. Fig. 9-b is the structural representation of carrier for expression of eukaryon pCHO1.0.

Figure 10 is the agarose gel electrophoresis figure that AT3002 fusion protease cuts qualification (HindIII, BamHI double digestion). Wherein, swimming lane 1 is after the expression vector p3xFLAG-CMV-13 double digestion containing AT3002 gene; Swimming lane 2 is 200bpDNAladder.

Figure 11 is the western blot figure of HLF cell expressing AT3002. Wherein, swimming lane 1 is pre-dyed albumen ladder; Swimming lane 2 and 3 is cell pyrolysis liquid and the cells and supernatant of HLF respectively; Swimming lane 4 and 5 is cell pyrolysis liquid and the cells and supernatant of HLF/AT3002 respectively.

Figure 12 is the western blot figure of expressing cho cell AT3002.

Figure 12 a is the western blot figure that after plasmid p3xFLAG-CMV-13-AT3002 proceeds to Chinese hamster ovary celI, AT3002 expresses. Wherein, swimming lane 1 is pre-dyed albumen ladder; Swimming lane 2 is the cell lysate supernatant (positive control) of HLF cell expressing AT3002; Swimming lane 3 is the cell lysate supernatant of expressing cho cell AT3002.

Figure 12 b is the western blot figure that after plasmid pCHO1.0-AT3002 proceeds to Chinese hamster ovary celI, AT3002 expresses. Wherein, swimming lane 1 is pre-dyed albumen ladder; Swimming lane 2 is CHO initiating cell culture supernatant (blank); Swimming lane 3 is the cell lysate supernatant of expressing cho cell AT3002.

Figure 13 is the anticancer experiment in vitro result (mtt assay) of HLF cell expressing AT3002 fusion rotein

Figure 13-a is that HLF cell expressing AT3002 fusion rotein is to the lethal experimental result of SW1116 colon cancer cell; Figure 13-b is that AT3002 fusion rotein is to the lethal experimental result of COLO205 colon cancer cell; Figure 13-c is that AT3002 fusion rotein is to the lethal experimental result of LS174T colon cancer cell; Figure 13-d is that AT3002 fusion rotein is to the lethal experimental result of T84 colon cancer cell; Figure 13-e is that AT3002 fusion rotein is to the lethal experimental result of LoVo colon cancer cell. Experimental result shows, above-mentioned five strain colorectal cancer cells are all had lethal effect by AT3002, and there is certain dose-effect relationship.

Figure 14 is the multimerization analysis (western blot figure) of HLF cell expressing AT3002. Wherein, swimming lane 1,3 is pre-dyed albumen ladder; Swimming lane 2,4 is AT3002 fusion rotein under reductive condition and non-reduced condition respectively. Illustrate that the AT3002 fusion rotein expressed in reconstitution cell exists mainly with the form of polymer.

Figure 15 is the Ca of HLF cell expressing AT3002 fusion rotein from different colon cancer cell²⁺Dependency Binding experiment (100x, immunocyte fluorescence). Figure is AT3002 and SW1116, COLO205, LS174T, T84, LoVo cell composition graphs at different conditions, result display AT3002 can with above-mentioned colon cancer cell specific combination, and combine there is Ca²⁺Dependency.

Figure 16 is the anticancer experiment in vitro result figure (mtt assay) of expressing cho cell AT3002 fusion rotein.

Figure 17 is the Ca of expressing cho cell AT3002 fusion rotein and COLO205 colon cancer cell²⁺Dependency Binding experiment (100x, immunocyte fluorescence).

Figure 18 is the microphotograph that HLF/AT3002 cell microcarrier is cultivated. Wherein, Figure 18-a and Figure 18-b be respectively 40x and 100x doubly under Photomicrograph.

Figure 19 is the electrophorogram before and after AT3002 protein purification or western blot figure.

Figure 19-a is the protein electrophoresis figure that AT3002 collects concentrated 10 times of supernatant. Wherein, swimming lane 1 is pre-dyed albumen ladder; Swimming lane 2 is that 10 times of concentrated AT3002 collect supernatant. Figure 19-b is the protein electrophoresis figure after AT3002 protein purification. Wherein, swimming lane 6 is pre-dyed albumen ladder; Swimming lane 1-5 and 7-11 is the collection liquid of different collection tube after collecting after AT3002 purifying; Swimming lane 12 wears liquid for stream. Figure 19-c is the western blot figure after AT3002 protein purification. Wherein, swimming lane 6 is pre-dyed albumen ladder; Swimming lane 1-5 and 7-11 is the collection liquid of different collection tube after collecting after AT3002 purifying; Swimming lane 12 wears liquid for stream.

Figure 20 is the tumor growth curve figure of HLF/AT3002 Carbazole alkaloid COLO205 colorectal carcinoma.

Figure 21 is the tumor weight statistical graph that HLF/AT3002 cell therapy COLO205 colorectal carcinoma tests each group.

Figure 22 is the tumour photo that HLF/AT3002 cell therapy COLO205 colorectal carcinoma tests each group.

Figure 23 is the mice with tumor photo that HLF/AT3002 cell therapy COLO205 colorectal carcinoma tests each group.

Figure 24 is tumor tissue section's colored graph that HLF/AT3002 cell therapy COLO205 colorectal carcinoma tests each group.

Figure 25 is the tumor growth curve figure of HLF/AT3002 Carbazole alkaloid LS174T colorectal carcinoma.

Figure 26 is the tumor weight statistical graph that HLF/AT3002 cell therapy LS174T colorectal carcinoma tests each group.

Figure 27 is the tumor growth curve figure that AT3002 fusion rotein suppresses transplanted tumor in nude mice growth.

Figure 28 is the statistical graph that AT3002 fusion rotein suppresses transplanted tumor in nude mice growth.

Figure 29 is that AT3002 fusion rotein is on the impact of transplanted tumor in nude mice survival time of mice.

Figure 30 is the computer aided design (CAD) figure of AT3132 fusion rotein.

Wherein, Figure 30-a is the structural simulation figure of AT3132 fusion rotein, G1-L177:TNF ��; E178-K192: connection peptides (EAAAK)₃; Q193-A538:DC-SIGN. Figure 30-b is the TNF �� structural simulation comparison in TNF �� monomer and AT3132 fusion rotein, illustrated blocks region representation TNF �� acceptor and TNF �� interaction region. Functional domain and the monomeric protein TNF �� of the TNF �� in fusion rotein do not have anything to change. As can be seen from the figure TNF �� almost overlaps completely with the TNF �� end of AT3132 fusion rotein. Therefore, it is also possible to speculate that the albumen tertiary structure of the TNF �� end in AT3132 fusion rotein and TNF �� monomer is still consistent, that is can speculate that the TNF �� end in AT3132 fusion rotein can still retain the biologic activity of TNF �� monomer. Figure 30-c is that AT3132 fusion rotein docks detail view with TNF �� acceptor, it is seen that the TNF �� in fusion rotein AT3132 and the combination between its acceptor are without space steric hindrance, it is possible to interact.

Figure 31 is the western blot figure of expressing cho cell AT3132 fusion rotein. Wherein, swimming lane 1 is the emiocytosis supernatant of CHO/AT3002 fusion rotein; Swimming lane 2 is pre-dyed albumen ladder; Swimming lane 3 is the Chinese hamster ovary celI culture supernatant (negative control group) of non-transgene; Swimming lane 4 is the cells and supernatant of CHO/AT3132.

Figure 32-a and Figure 32-b are the biologic activity detected result of AT3132 fusion rotein. Wherein, Figure 32-a is AT3132 fusion rotein to the lethal test-results of COLO205 colon cancer cell, and result shows, and compared with comparison TNF ��, AT3132 and A3002 equally can effectively kill and wound colorectal cancer cells; Figure 32-b is AT3132 fusion rotein and the Ca of COLO205 cell²⁺Dependency Binding experiment (100x, immunocyte fluorescence). Result shows, and AT3132 is combined with colorectal cancer cells has Ca²⁺Dependency.

Embodiment

Now the present invention is described with the following Examples. There is provided these embodiments only for illustrative purposes, the invention is not restricted to these embodiments, but comprise obviously by being changed that instruction provided herein produces.

The gene design of embodiment 1 fusion rotein

One, connection peptides screening

First the present invention chooses connection peptides G10 as the connection peptides of TNF �� and DC-SIGN.

Application SYFPEITHI, MH2pred, ANNpred software, and adopt its default parameters to calculate respectively, calculate the T cell immunogenicity mark of G10 is: 5(best result), 0,0, application Bcepred, ABCpred calculate B cell immunogenicity mark: 0,0.51, the T cell and the B cell immunogenicity mark that calculate after normalization method respectively are 0.0 and 0.51, all belong to weak immunogenicity.

Two, albumen model construction

The present invention preferably adopts DC-SIGN as recognition function territory, GenBank corresponding to DC-SIGN is numbered M98457, corresponding albumen is numbered AAF77072 (Fig. 1), the full name of DC-SIGN be DC surface specific cell between adhesion factor 3 in conjunction with nonconformity element molecule. The amino acid of DC-SIGN used in the present invention is 59-404. The present invention preferably adopts TNF �� as action function territory, is numbered X01394 in the GenBank corresponding to TNF ��, and corresponding albumen is numbered NP_000585 (Fig. 1), and full name is TNFa lpha. TNF �� in the present invention amino acid used is 57-233.

Amino acid (representing with the single-letter) sequence of TNF ��, G10 and DC-SIGN being connected in order, form new fusion rotein aminoacid sequence, new fusion rotein amino acid length is 533 amino acid, and this sequence is as shown in SEQIDNO:2. For stating conveniently, unified by this fusion rotein TNF ��-G10-DC-SIGN(or TNF ��/DC-SIGN in the application) it is denoted as " AT3002 ".

The TemplateIdentification that DC-SIGN and TNF �� apply SWISS-MODEL software respectively finds template, and the template (PDB numbering 1A8M, B chain) that TNF �� obtains, has all had three-dimensional structure from 82-233 amino acid, it is necessary to build 57-81 amino acid; From the existing three-dimensional structure (PDB numbering 2IT5) of 253 to 384 amino acid in DC-SIGN aminoacid sequence, it is necessary to build 59-252 and 384-404 amino acid three-dimensional structure. For modeller modeling after the molecule of TNF �� and DC-SIGN template removal water molecules and other Non-covalent binding.

Adopt Modeller program to carry out monomeric protein homology model structure, comprise framework construction, ring texture simulation and side chain and add. DC-SIGN 3 d structure model has 346 amino acid, and TNF �� 3 d structure model has 177 amino acid. After initial model has built, application molecular dynamics carries out system energy optimization and molecular dynamics simulation, and the significant parameter of Sander program used is: Maxcyc=2500, Ncyc=1000, Cutoff=10. Molecular dynamics simulation is applied some assessment processes (StructuralAnalysisandVerificationServer) and is carried out model evaluation after completing, adjust for the parameter not meeting stereochemistry requirement pointed out in assessment.

Spatially carry out rotating and translating by the 3 d structure model of DC-SIGN and TNF ��, make both most major axis be similar to parallel and distance and be aboutThese two three-dimensional structures are write same Three dimensional structure files, carrys out construction of fusion protein 3 d structure model (Fig. 2) with this as new template application Modeller program. Fusion rotein model construction is applied molecular dynamics and is carried out system energy optimization and molecular dynamics simulation after completing, the significant parameter of Sander program used is: Maxcyc=2500, Ncyc=1000, Cutoff=10. The model of fusion rotein is assessed, the parameter not meeting stereochemistry requirement pointed out in assessment is adjusted.

Three, molecular docking

TNF �� belongs to the member of tumor necrosis factor superfamily, and this superfamily member is numerous, but has a segment length about 150 amino acid whose sequences, (TNFhomeodomains, THD), and this sequence is the conservative skeleton containing aromatic series and hydrophobic residue. THD in different TNF family protein structure has almost completely identical space folding configuration, and the albumen with tripolymer is formed with substantial connection. The acceptor of the correspondence of TNF family is also very many, and characteristic structural is the structural domain (CRDs) that its extracellular domain is rich in halfcystine, usually has 6 cysteine residues to participate in forming three disulfide linkage. From existing achievement in research, the acceptor of TNF family and the interaction pattern of part are diversified, but are first that TNF family protein molecule forms tripolymer, and then its part and acceptor form dimer pattern. At TNF �� and TNF ��-R(PDB numbering 1TNR), TRAIL and TRAIL-R2(PDB numbering 1D4V), TNF �� and TNF ��-R (PDB numbering 3ALQ) interact with 3:3 binding pattern output. The present invention is using TNF �� and TNF ��-R (PDB numbering 3ALQ) interaction pattern as the starting point of molecular docking. Adopt the online software of RosettaDock (http://rosettadock.graylab.jhu.edu) that TNF �� and TNF ��-R are carried out molecular docking. In the same way the TNF �� in TNF ��-G10-DC-SIGN fusion rotein and TNF ��-R should be carried out molecular docking (Fig. 3).

Four, structure alignment and marking

DC-SIGN in DC-SIGN and TNF �� monomer 3 d structure model and fusion rotein and TNF �� structural domain are carried out structure alignment (Fig. 4) by the Superpose program of application CCP4 software package, consider that DC-SIGN and TNF �� have a large amount of ring structure, molecular dynamics simulation can occur bigger change, structure alignment result can be had a huge impact, so selecting to safeguard that the skeleton structure of its function carries out structure alignment. The part selecting the amino acid whose skeleton structure of 202-322 corresponding with fusion rotein DC-SIGN monomer carries out structure alignment, and gained RMSD value isCorresponding quantization parameter (Tm-score) is 0.991; Selecting 33-177 amino acid to carry out structure alignment TNF �� monomer, gained RMSD value isCorresponding quantization parameter (Tm-score) is 0.988, two mean values is 0.9895. In two monomers and fusion rotein, DC-SIGN and TNF �� are by the molecular docking result obtained, and compare the molecular docking result of acceptor and part before merging and the molecular docking result of acceptor and part after merging, and the structure calculating TNF �� changes, and RMSD value isCorresponding quantization parameter (Tm-score) is 0.976. In addition, the residue distance of the TNF �� in fusion rotein and its ligand interaction existsBetween, it is possible to form more stable hydrogen bond. As can be seen here, simulation result according to computer can show that the space structure of each monomer does not have obvious difference under the space structure of the recognition function territory DC-SIGN in fusion rotein and action function territory TNF �� and native state, and the TNF �� in fusion rotein still saves its biological activity, it is possible to the efficient and concurrent looks mutual effect of its receptors bind.

The gene optimization of embodiment 2AT3002 fusion rotein

1. codon optimized

In order to express this AT3002 fusion rotein, it has also been carried out codon optimized by applicant, the optimization of mRNA structural modifications and translation initiation site. Obtain gene order as shown in SEQIDNO:1. Gene pairs before and after optimizing is than as shown in Figure 1.

Here is that AT3002 gene carries out each parameter comparison in codon optimized front and back is as follows:

1. codon adaptation index (CodonAdaptationIndex, CAI)

By Fig. 6-a it will be seen that before codon do not optimize, AT3002 gene codon adaptation index (CAI) in mammalian cell is 0.84. By Fig. 6-b it will be seen that by after codon optimized so that the AT3002 gene of the present invention CAI index in mammalian cell is 0.85. Be considered to this gene during usual CAI=1 is optimal efficient expression status in this expression system, CAI index is more low shows that this gene expression level in this host is more poor, therefore can find out have passed through codon optimized after the gene order that obtains can improve the expression level of AT3002 gene in mammalian cell.

2. optimal codon uses frequency (FrequencyofOptimalCodons, FOP)

By Fig. 7-a it will be seen that based on mammalian cell expression vector, before codon is not optimized, the low utilization ratio codon of AT3002 gene order occurs that per-cent is 5%. This gene not being optimized contains series connection rare codon, and these codons may reduce translation efficiency, even can dismiss translation assembling thing. By Fig. 7-b it will be seen that by after codon optimized, the AT3002 gene of the present invention occurs that in mammalian cell system the frequency of low utilization ratio codon is 1%.

3.GC base contents (GCcurve)

G/C content ideal distribution region is 30%-70%, and any peak of the appearance outside this region all can affect to some extent transcribes and translation efficiency. Contrast from the GC base average content distributed areas figure of the AT3002 gene of Fig. 8-a, Fig. 8-b, it is 57.25% by Fig. 8-a shows in AT3002 gene GC base average content before optimization, by Fig. 8-b shows finally be optimized after the GC base average content of heavy AT3002 gene be 54.57%, be more conducive to the expression of AT3002 gene.

4. before and after optimizing, cis-acting elements situation is as follows:

5. before and after optimizing, the palindrome and tumor-necrosis factor glycoproteins situation are as follows:

The gene chemical synthesis of embodiment 3AT3002 fusion rotein and expression vector establishment

AT3002 fusion rotein full genome after above-mentioned optimization is carried out synthetic, and adds the restriction enzyme site of HindIII and BamHI at the two ends of antigen-4 fusion protein gene total length respectively. Introduce terminator codon site, to prevent the expression of FLAG label affects the character of fusion rotein on expression vector after BamHI site simultaneously. Antigen-4 fusion protein gene is inserted in p3xFLAG-CMV-13 plasmid (Fig. 9 a, plasmid is purchased from Sigma company) by above-mentioned two restriction enzyme sites, obtains one and preserves plasmid for a long time, is designated as p3xFLAG-CMV-13-AT3002 plasmid.

AT3002 fusion rotein full genome after above-mentioned optimization is carried out synthetic, and adds the restriction enzyme site of Avr II and BstZ171 at the two ends of antigen-4 fusion protein gene total length respectively. Antigen-4 fusion protein gene is inserted in pCHO1.0 plasmid (Fig. 9 b, plasmid is purchased from Invitrogen company) by above-mentioned two restriction enzyme sites, obtains one and preserves plasmid for a long time, is designated as pCHO1.0-AT3002 plasmid.

The expression of embodiment 4AT3002 fusion rotein in HLF cell and qualification

One, recombinant expression vector electricity transforms HLF cell

The carrier for expression of eukaryon p3xFLAG-CMV-13-AT3002 above-mentioned synthesis obtained proceeds to bacillus coli DH 5 alpha (purchased from Tian Gen biochemical technology company limited), cultivates amplification, extracting plasmid. Plasmid carries out digestion verification (Figure 10), and digestion verification result shows, and the gene size of above-mentioned AT3002 is consistent with expection. Plasmid electrotransformation after amplification proceeds to HLF cell (purchased from JCRBcellbank), and specifically operation is as follows:

1., by HLF cell cultures to the degree of collecting of 60%-80%, after pancreatin (purchased from Amersco) digestion, it is resuspended in former training

In nutrient solution;

2.1000rpm, the centrifugal 5-7min of room temperature, abandons supernatant, resuspended with 2mLPBS damping fluid;

3. electricity conversion condition: electricity turns voltage 110V, and electricity turns time 25ms, adds cell culture fluid after electric shock immediately;

Be replaced by containing 1%G418(purchased from Sigma after 4.12h) screening cell culture fluid.

Two, the preparation of positive colony protein sample

When the cell after electricity transforms grows to confluent culture bottle 80-100% concentration, change ASF104 serum free medium (purchased from Ajinomoto, Japan). After 2-3d, collect serum free medium, enrichment medium. Cell scraper is resuspended to the general lysis buffer (proportioning: 25mMTris, 150mMNaCl, 2mMEDTA, 1%NP40,5%Glycine, pH7.4 of 500uL after collecting. With front adding 1% proteinase inhibitor) at 4 DEG C of cracking 1h.

Three, the expression of immunoblotting qualification fusion rotein

It is the anti-human DC-SIGN antibody (purchased from R&D) of mouse that immunoblotting detects primary antibodie used, and two resist for HRP-rabbit anti-mouse antibody (purchased from Sigma). The theoretical molecular of AT3002 is 60kd, by the SDS-PAGE glue separation of the substratum after concentrated in step 2 and cell pyrolysis liquid 10%. As can be seen from Figure 11, in the born of the same parents of HLF/AT3002, detect the band of specificity with extracellular products, and the empty cell controls of the HLF of control group 2,3 swimming lane does not detect the expression of AT3002. AT3002 in born of the same parents and the AT3002 molecular weight of secreting, expressing have the difference of about 10kd. Wanting in the AT3002 molecular weight ratio born of the same parents of secretion is big, and this may be relevant with the posttranslational modification processing of fusion rotein.

The expression of embodiment 5AT3002 fusion rotein in Chinese hamster ovary celI and qualification

Adopt the method for embodiment 4 that plasmid p3xFLAG-CMV-13-AT3002 and pCHO1.0-AT3002 in embodiment 3 is proceeded to Chinese hamster ovary celI (purchased from ATCC) respectively and culture expression, carry out expression identification with immunoblotting. As shown in figure 12, AT3002 fusion rotein also can be expressed in Chinese hamster ovary celI. CHO is recombinant protein general in the world and therapeutic monoclonal antibodies expression cell line. The expression of AT3002 in Chinese hamster ovary celI has also implied that AT3002 fusion rotein possesses the potentiality of industrialization.

The biologic activity detection of embodiment 6HLF cell expressing AT3002 fusion rotein

One, the colorectal cancer cells fragmentation test of fusion rotein

This experiment chooses the colon carcinoma cell line COLO205(of sugar chain specificity overexpression purchased from JCRBcellbank), SW1116(is purchased from JCRBcellbank), LS174T(is purchased from Chinese Academy of Sciences's Shanghai cell bank), T84(is purchased from Chinese Academy of Sciences's Shanghai cell bank) and LoVo(purchased from Chinese Academy of Sciences's Shanghai cell bank) study the anti tumor activity in vitro of fusion rotein. Anti-tumor activity detection adopts mtt assay. The HLF/AT3002 expressing fusion protein cell strain cultivated with ASF104 serum free medium (purchased from Ajinomoto, Japan) expresses supernatant, frozen, and standby MTT experiment is used. Above-mentioned colorectal cancer cells is cultivated on 24 orifice plates according to the concentration in 50000/hole, adds fusion rotein culture supernatant to be detected or TNF �� standard substance (purchased from ProSpec, Israel) simultaneously, adds the Ca of 10mM simultaneously²⁺. After 48h, adding MTT(final concentration is 500ug/mL), to continue to hatch 4h, terminate cultivating, careful suction abandons culture supernatant in hole. For suspension cell need centrifugal after again inhale abandon culture supernatant in hole. Every hole adds 400uLDMSO, and vibration 10min, makes crystallisate fully melt. Selecting 490nm wavelength, measure each hole absorbance value on enzyme linked immunological monitor, the more big explanation cell viability of light absorption value is more strong, and the more little tumour cell of light absorption value is suppressed or is killed more many.

Figure 13-a to 13-e is that AT3002 fusion rotein is to the lethal test-results figure of this five strains colon cancer cell respectively. As seen from the figure, SW1116, COLO205, LS174T, T84 and LoVo are had certain fragmentation effect by AT3002. Wherein, the fragmentation effect of COLO205, LS174T and T84 is dose-dependently by AT3002. These experimental results show, AT3002 fusion rotein still remains the tumor cell killing potential of TNF ��.

Two, the polymer of fusion rotein is formed and analyzes

The multimerization analysis non-denatured protein electrophoresis detection of fusion rotein, operation steps is shown in (" fine works protein science experiment guide " 2010 editions, the work such as J.E. Ke Lingen, P303-P307). Figure 14 is the multimerization analysis of AT3002 fusion rotein. As can be seen from the figure, the fusion protein molecule amount of non denatured is much larger than the fusion rotein of sex change, and this illustrates that fusion rotein there occurs multimerization, causes molecular weight to increase. And forming one of the characteristic that polymer is C type lectin, the multimerization of fusion rotein is likely caused by the multimerization of C type lectin. The fusion rotein of multimerization at the short tumor death signal of local enhancement, can increase its anti-tumor activity.

Three, the sugared chain combination ability detection of fusion rotein

The method that this experiment is tested with cellular immunofluorescence detects. Sketch as follows: 24 orifice plates or 96 orifice plate cell climbing sheets (COLO205, SW1116); Inhale substratum supernatant, TBS rinsing 3 times; 4%PFA(is purchased from Sigma) fixing 20min, TBS wash three times; Close 1h, TBS with the serum (or 5% skimmed milk) of two anti-identical hosts and wash three times; The cell handled well and fusion rotein secretion supernatant are hatched 1h(and are added 10mMCa²⁺, 10mMEDTA or 10mM seminose), primary antibodie (the anti-human DC-SIGN antibody of mouse, purchased from R&D, with 2.5% skimmed milk dilution) 4 DEG C is spent the night, or room temperature 1h, and TBS washes three times; Anti-(Alexa-514 rabbit against murine traget antibody, dilute with TBS) the room temperature 1h(lucifuge of fluorescent mark two), or 37 DEG C of 45min, PBS wash three times (lucifuge); Envelope sheet, fluorescence microscopy Microscopic observation.

Complete have active fusion rotein except can killing tumor cell, also should have can target to the ability in conjunction with tumour cell. This is tested by cellular immunofluorescence, investigates the binding ability of fusion rotein and colon cancer cell SW1116, COLO205, LS174T, T84 and LoVo. Figure 15 is the immunofluorescence micrograph of AT3002 and colon cancer cell. Lectin has Ca with the combination of sugar chain²⁺Dependency. As can be seen from the figure, 10mMCa is being added²⁺After, AT3002 can be combined by strain colon cancer cell a few with this, has stronger fluorescent signal. But then inhibit this kind to combine after adding 10mMEDTA, this is because EDTA can chelating Ca²⁺, thus prevent the combination of C type lectin and colorectal cancer cells. Meanwhile, the seminose (mannose) adding 50mM competitively in conjunction with fusion rotein and colorectal cancer cells binding site, can cause fusion rotein cannot be combined with colorectal cancer cells. In addition, 10mMCa is not being added²⁺Control group also can see having strong fluorescence to combine, this illustrates the Ca of physiological concentration²⁺Also the needs that AT3002 is combined can be met with colorectal cancer cells.

Targeting is a ring the most important in fusion rotein Evaluation of Functional. Only fusion rotein has targeting healthy tissues and cell just can be avoided from the short impact of molecule of dying of withering, and maximum degree could reduce the side effect of pro apoptotic protein, is also one of problem of paying close attention to the most in medical judgement. In the fusion rotein colorectal cancer cells killing experiments of the present embodiment, AT3002 not only has good anti-tumor activity, also has the binding ability of sugar chain specificity simultaneously.

The biologic activity detection of embodiment 7CHO cell expressing AT3002 fusion rotein

Adopt the method for embodiment 6 that the AT3002 fusion rotein that CHO in embodiment 5 expresses is carried out Activity determination. Figure 16 examines the AT3002 of CHO expression to the kill capability of COLO205 cell. As seen from the figure, AT3002 group is relative to control group, and cell viability decline is a lot, and it is active that this illustrates that the AT3002 fusion rotein of expressing cho cell has good tumor cytotoxicity equally.

The tumour cell binding activities research of expressing cho cell AT3002 can also be found out (Figure 17), adding Ca²⁺After EDTA, inhibit AT3002 albumen and the binding ability of COLO205 cell. Meanwhile, the binding ability of AT3002 fusion rotein is expressed by contrast HLF, it can be seen that the AT3002 that CHO expresses has fluorescence developing more clearly. This illustrates that the combination of the AT3002 that CHO expresses and tumour cell has higher specificity, and keying action has Ca²⁺Dependency. The enlarged culturing of embodiment 8HLF/AT3002 expressing fusion protein cell strain

HLF cell is attached cell, cannot carry out suspension culture. The present invention has carried out the enlarged culturing of HLF cell by the method for microcarrier suspension culture. Cytodex1 microcarrier is purchased from GE company. Getting appropriate microcarrier and be dissolved in PBS and sterilizing, add MEM substratum (biological purchased from clear big sky one), add the microcarrier of 3-5g/L in the shaking flask of sterilizing, cell inoculum size is 100000 cell/mL, changes ASF104 serum free medium after cultivating 3d. Supernatant is collected, for the separation and purification of fusion rotein after continuing to cultivate 2-3d. Figure 18-a, Figure 18-b are the Photomicrograph that microcarrier cultivates HLF/AT3002 cell. As shown in the figure, HLF cell can on microcarrier normal growth. This have found suitable method for HLF/AT3002 enlarged culturing (suspension culture). The separation and purification of embodiment 9AT3002 fusion rotein

AT3002 fusion rotein mannosans-agarose plugs affinitive layer purification. Sample pretreatment:

1,2.6L supernatant adds 10x binding buffer liquid (proportioning: 10mMTris, 100mMNaCl, 10mMCaCl₂, pH7.4) to cumulative volume be 3L, mixed even;

2, the centrifugal 10min of 12000rpm, removes cell and cell debris;

3,0.45um membrane filtration.

Specific experiment step:

1, Homogeneous phase mixing filler, absorption of spending the night in 1x binding buffer liquid;

2, with the binding buffer liquid balance chromatography column being greater than 10 column volumes;

3, sample is according to 1-4mL/min flow velocity loading;

4, wash-out: elution buffer (proportioning: 10mMTris, 100mMNaCl, 10mMEDTA, PH7.4), collects elutriant. Protein SDS-PAGE electrophoresis and immunoblotting detection purity of protein.

Figure 19 is the protein electrophoresis figure of the AT3002 before and after purifying, from picture, it does not have the assorted band of the AT3002 fusion rotein of purifying is many, and the substantially not assorted band of AT3002 after purifying, illustrate that purification effect is better.

The HLF cell of embodiment 10 expressed fusion protein AT3002 is to the cell therapy effect of COLO205 tumour

The present invention chooses transplanted tumor in nude mice model to investigate the anti-tumor activity of fusion rotein. By by subcutaneous to naked mouse to the HLF cell of expressed fusion protein and colon cancer cell COLO205 combined inoculation, investigating the restraining effect that colorectal carcinoma COLO205 Transplanted cells knurl is grown by HLF emiocytosis fusion rotein. Specific experiment scheme is as follows: by cultured cell in vitro by 10⁸/ mL concentration re-suspended cell, experiment is divided into model group (COLO205), AT3002 group (COLO205+HLF/AT3002) and two control group (COLO205+HLF/DC-SIGN; COLO205+HLF/X-DC-SIGN), only often organize 9-10. Wherein, the cell of COLO205+HLF/DC-SIGN control group inoculation only expresses target to end structure territory DC-SIGN; The cell of COLO205+HLF/X-DC-SIGN control group inoculation is express target to the HLF cell of end structure territory DC-SIGN and unrelated protein (human serum albumin) amalgamation and expression. HLF/DC-SIGN and HLF/X-DC-SIGN cell strain is all prepared according to the method for embodiment 4, is inserted in p3xFLAG-CMV-13 carrier by goal gene DC-SIGN or X-DC-SIGN, and electricity transforms HLF cell, obtains positive colony cell strain. According to grouping cell equal proportion is mixed, subcutaneous injection 6-8 week age female naked mouse, build transplanted tumor in nude mice model. Naked mouse is purchased from Shanghai Si Laike animal center, and adaptability raises inoculated tumour after 5d. After having inoculated, the growing state of monitoring tumour, the size of tumour calculates according to length �� wide �� wide/2. After three weeks, naked mouse is put to death, and peels off tumour, weighs tumor weight. In addition, tumor tissues has also been carried out paraffin section and immunochemistry tissue staining by this experiment, detects the expression of tumor tissues place AT3002. Organization embedding and paraffin section complete in Zhenjiang First People's Hospital. The dyeing of tumor tissue section is with reference to conventional dyeing process (" diagnosis immunohistochemistry " 2011 editions, Ji little Long writes, P14-P39), and the primary antibodie hatched is DC-SIGN antibody (purchased from R&D), and two resist for HRP-rabbit anti-mouse antibody (purchased from Sigma). Only chromogenic substrate takes TMB colour developing liquid (purchased from AMRESCO) different from reference method.

Figure 20 is the growth curve chart that cell therapy is respectively organized. As can be seen from the figure, the naked mouse tumor growth of the administration group being vaccinated with HLF/AT3002 obtains obvious suppression, almost can't detect the existence of tumour to the later stage. And the tumor growth of model group and two control groups is clearly. The statistics of naked mouse tumor weight be have also been obtained same result (Figure 21). Figure 22 and Figure 23 is the tumour photo of each group, also reflects the situation of each group of tumor growth. Figure 22 is the photo peeling off tumour, and Figure 23 is the mice with tumor photo before tumour is peeled off. As can be seen from the figure, the tumour of model group and control group is bigger than AT3002 group volume, and this illustrates that the tumor growth of AT3002 group obtains suppression. The section of tumor tissues has also been carried out dye (Figure 24) by this experiment. Coloration result shows, and color reaction can occur at the tumor tissues position of AT3002 group, shows that AT3002 goes out to express and enrichment in tumour; And control group COLO205 does not develop the color. This AT3002 really illustrated can the growth being combined also inhibition tumor cell with tumor tissues of specificity.

Embodiment 11 expressed fusion protein HLF cell is to the cell therapy effect of LS174T tumour

The method of embodiment 10 is adopted the anti-LS174T colorectal carcinoma effect of HLF/AT3002 to be studied. Specific experiment scheme is identical with embodiment 10, is divided into two groups: negative control group, HLF+COLO205; AT3002 group, HLF/AT3002+COLO205. Figure 25 is the growth curve chart of two groups of tumours, it can be seen that the tumor growth rate of AT3002 group is compared control group and received obvious suppression. The weight of two groups of tumours has been carried out statistics and have also been obtained same result by Figure 26.

The anti-tumor in vivo activity rating of embodiment 12AT3002 fusion rotein

The present invention chooses transplanted tumor in nude mice model to investigate the anti-tumor activity of AT3002. With the female naked mouse in COLO205 cell subcutaneous injection 6-8 age in week, build transplanted tumor in nude mice model, by the AT3002 naked mouse of fusion rotein abdominal injection after purifying, observe tumor growth situation. Concrete operation is as follows: the COLO205 cell of vitro culture, purchased from Shanghai Si Laike animal center, after adaptability raises 5d, is pressed 10 by naked mouse⁸/ mL concentration is resuspended, often only naked mouse subcutaneous vaccination 10⁷Individual COLO205 cell (200uL). After 7d, when tumour grows to diameter 6-7mm, reject the naked mouse that small part does not become knurl, random point three groups (n=8 or 9). Experiment is divided into control group (TBS-Ca damping fluid), small dose group (40ug/kg), heavy dose of group (400ug/kg) three groups. Mouse intraperitoneal injection every day after grouping, successive administration 10d. Monitor the growing state of tumour every day, and add up its lifetime. The size of tumour calculates according to length �� wide �� wide/2. Tumor growth rate calculation formula: (the whole opisthosoma of tumour amasss-tumour original volume) �� 100%/tumour original volume. Tumor control rate calculation formula: (the whole opisthosoma of the long-pending administration group tumour of the whole opisthosoma of control group tumour amasss) whole opisthosoma of �� 100%/control group tumour amasss.

Experimental result shows, compared with control group, the growth of high and low dose group transplanted tumor all receives suppression (Figure 27, Figure 28), and the tumor control rate of small dose group and heavy dose of group reaches 45.0% and 57.7% respectively, it is seen that inhibiting rate and dosage present obvious dose-effect relationship. Meanwhile, the lifetime of mice with tumor has also been added up by this experiment, statistical result showed (Figure 29), and after 50 days, naked mouse substantially all deads of control group, compare control group, and there has been significant raising the lifetime of AT3002 protein medicine-feeding group. It thus is seen that AT3002 can obviously suppress and killing tumor cell, it is possible to effectively extend the lifetime of mice with tumor.

The design of embodiment 13AT3132 fusion rotein and preliminary active assessment

The present invention also have chosen the connection peptides belonging to equally and having better molecular flexibility in addition: (EAAAK)₃, it can form the higher structure of spirrillum, the protein structure domain being connected with its two ends is fully unfolded and comes. The present invention also studied (EAAAK) equally₃As the fusion rotein of TNF �� and DC-SIGN connection peptides. For stating conveniently, unified by fusion rotein TNF ��-(EAAAK) in the application₃-DC-SIGN is denoted as " AT3132 ", and its aminoacid sequence is as shown in SEQIDNO:4.

The present invention also simulates the higher structure (Figure 30-a) of AT3132 further by the method for embodiment 1. The TNF �� albumen of the AT3132 fusion rotein of simulation and monomer is carried out structure alignment (Figure 30-b), it can be seen that the N end of AT3132 almost overlaps completely with TNF ��, illustrate that both structure changes are small. The simulation test (Figure 30-b, Figure 30-c) of molecular docking shows that AT3132 is consistent with docking of monomer whose albumen with the docking of TNF �� R, thus can prove that the structure of AT3132 fusion rotein does not affect the activity of its TNF �� structural domain further.

The gene of fusion rotein is optimized (nucleotide sequence after optimization is as shown in SEQIDNO:3) by the method for present invention embodiment 2, and according to the method for embodiment 5, the AT3132 gene after optimization is proceeded to Chinese hamster ovary celI and carries out expressing and identifying. Figure 31 is the immunoblotting qualification figure of AT3132 albumen. As can be seen from the figure, the molecular size of AT3132 is close with AT3002; The brightness of band is also identical with A3002, illustrates that both expression amounts are also more or less the same. In order to verify the biologic activity of AT3132, the present invention takes the technique study of embodiment 6 AT3132 to the kill capability of COLO205 cell and binding ability. Test-results (Figure 32-a, Figure 32-b) shows that AT3132 can suppress the growth of colon cancer cell COLO205; AT3132 has Ca too²⁺The binding ability of dependency, this kind of binding ability can be suppressed by EDTA and seminose (mannose). The experimental result of the present invention shows to adopt (EAAAK)₃The TNF �� of connection peptides is same with DC-SIGN fusion rotein has good biologic activity.