CN108794619A - A kind of high affine PD-1 protein mutants - Google Patents

The invention discloses a kind of high affine PD-1 protein mutants, by being mutated four amino acid sites of wild type PD-1：M70I,S87W,A129H,K135M；The PD-1 protein mutants with more high-affinity are obtained, not only can significantly raise T cell activation and cytokine secretion, while dosage can also be reduced, reduce treatment cost.The present invention also provides the purposes of the mutant or its preparation in preparing treatment, preventing or alleviating the drugs such as tumor-related illness.

一种高亲和PD-1蛋白突变体A high-affinity PD-1 protein mutant

技术领域technical field

本发明涉及分子生物领域，尤其涉及一种高亲和PD-1蛋白突变体，蛋白的结构、对应氨基酸、对应基因序列、制备方法及应用。The present invention relates to the field of molecular biology, in particular to a high-affinity PD-1 protein mutant, protein structure, corresponding amino acid, corresponding gene sequence, preparation method and application.

背景技术Background technique

程序性细胞死亡蛋白(PD-1)是一种具有268个氨基酸的I型膜蛋白，属于CD28家族成员，其基因位于2号染色体上(The EMBO Journal(1992)，vol.11，issue 11，p.3887-3895)，其中人的PD-1cDNA由如EMBL/GenBank Acc.No.NM_005018所示的碱基序列组成。据报告，PD-1蛋白主要表达于CD4⁺CD8⁺的T细胞表面上(International Immunology(1996)，vol.18，issue5，p.773-780.，J.Experimentaled.(2000)，vol.191，issue 5，p.891-898.)。在骨髓细胞中也观察到PD-1的表达，包括由抗原受体刺激活化的T细胞或B淋巴细胞或活化的巨噬细胞表面(International Immunology(1996)，vol.18，issue5，p.765-772.)。Programmed cell death protein (PD-1) is a type I membrane protein with 268 amino acids, which belongs to CD28 family members, and its gene is located on chromosome 2 (The EMBO Journal (1992), vol.11, issue 11, p.3887-3895), wherein the human PD-1 cDNA consists of the base sequence shown in EMBL/GenBank Acc.No.NM_005018. It is reported that PD-1 protein is mainly expressed on the surface of CD4 ⁺ CD8 ⁺ T cells (International Immunology (1996), vol.18, issue5, p.773-780., J. Experimentaled. (2000), vol.191 , issue 5, p.891-898.). The expression of PD-1 is also observed in bone marrow cells, including the surface of activated T cells or B lymphocytes or activated macrophages stimulated by antigen receptors (International Immunology (1996), vol.18, issue5, p.765 -772.).

PD-1是一种免疫抑制性分子，它的两个糖蛋白配体已得到鉴定，即PD-L1和PD-L2，其被证实与PD-1结合后能够传递免疫抑制性信号，下调T细胞活化和细胞因子分泌，促进肿瘤逃避免疫系统的监控与杀伤(Freeman et al.(2000)J.Exp.Med.192:1027-34；Latchmanet al.(2001)Nat.Immunol.2:261-8；Carter et al.(2002)Eur.J.Immunol.32:634-43；Ohigashi et al.(2005)Clin.Cancer Res.11:2947-53)。PD-1 is an immunosuppressive molecule, and its two glycoprotein ligands have been identified, namely PD-L1 and PD-L2, which have been confirmed to transmit immunosuppressive signals after binding to PD-1 and downregulate T Cell activation and cytokine secretion promote tumor evasion from immune system monitoring and killing (Freeman et al. (2000) J. Exp. Med. 192:1027-34; Latchman et al. (2001) Nat. Immunol. 2: 261- 8; Carter et al. (2002) Eur. J. Immunol. 32:634-43; Ohigashi et al. (2005) Clin. Cancer Res. 11:2947-53).

阻断PD-1和PD-L1相互作用成为治疗肿瘤的一条有效途径(Sharma and Allison2015)。目前已知的针对PD-1/PD-L1相互作用的抗肿瘤药物为单克隆抗体药物。然后单抗药物具有免疫原性，个体差异性以及适应症有限等先天局限性，因此设计高亲和PD-1突变体来作为体内PD-1/PD-L1通路的竞争性抑制剂对于调控PD-1通路具有好的应用前景，不但可以恢复T细胞活化和提高细胞因子分泌水平，同时还有可能用于分子探针来检测肿瘤生长情况(Maute，Gordon et al.2015，Abdin，Zaher et al.2018)。Blocking the interaction of PD-1 and PD-L1 has become an effective way to treat tumors (Sharma and Allison2015). Currently known anti-tumor drugs targeting PD-1/PD-L1 interaction are monoclonal antibody drugs. However, monoclonal antibody drugs have inherent limitations such as immunogenicity, individual differences, and limited indications, so high-affinity PD-1 mutants are designed as competitive inhibitors of the PD-1/PD-L1 pathway in vivo to regulate PD. The -1 pathway has good application prospects, not only can restore T cell activation and increase cytokine secretion levels, but also may be used for molecular probes to detect tumor growth (Maute, Gordon et al.2015, Abdin, Zaher et al .2018).

发明内容Contents of the invention

基于背景技术存在的技术问题，本发明提出了一种高亲和PD-1蛋白突变体。Based on the technical problems in the background technology, the present invention proposes a high-affinity PD-1 protein mutant.

本发明的技术方案如下：Technical scheme of the present invention is as follows:

一种高亲和PD-1蛋白突变体，其氨基酸序列为以下组中的任意一个；A high-affinity PD-1 protein mutant, the amino acid sequence of which is any one of the following groups;

A、如SEQ ID NO：1或3或5或7中的任一所示氨基酸序列；A. An amino acid sequence as shown in any one of SEQ ID NO: 1 or 3 or 5 or 7;

B、如SEQ ID NO：1或3或5或7中的任一所示氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸所得功能相同或相似的氨基酸序列。B. The functionally identical or similar amino acid sequence obtained by substitution and/or deletion and/or addition of one or several amino acids in the amino acid sequence shown in any one of SEQ ID NO: 1 or 3 or 5 or 7.

一种高亲和PD-1蛋白突变体，其基因序列为以下组中的任意一个；A high-affinity PD-1 protein mutant whose gene sequence is any one of the following groups;

A、如SEQ ID NO：2或4或6或8中的任一所示基因序列；A. Gene sequence as shown in any one of SEQ ID NO: 2 or 4 or 6 or 8;

B、如SEQ ID NO：2或4或6或8中的任一所示基因序列中经过取代和/或缺失和/或添加一个或几个基因所得功能相同或相似的基因序列。B. A gene sequence with the same or similar function obtained by substitution and/or deletion and/or addition of one or several genes in the gene sequence shown in any one of SEQ ID NO: 2 or 4 or 6 or 8.

一种高亲和PD-1蛋白突变体，该蛋白质选自野生型PD-1的四个氨基酸位点突变：M70I、S87W、A129H、K135M。A high-affinity PD-1 protein mutant, which is selected from four amino acid site mutations of wild-type PD-1: M70I, S87W, A129H, and K135M.

以上的PD-1蛋白突变体与真核蛋白hPD-L1-Fc具有更好的高亲和。The above PD-1 protein mutants have better high affinity with eukaryotic protein hPD-L1-Fc.

一种制剂，含有上述的PD-1蛋白突变体。A preparation comprising the above mutant PD-1 protein.

该PD-1蛋白突变体或其制剂在制备治疗、预防、或者缓解肿瘤相关疾病等药物中的用途。Use of the PD-1 protein mutant or its preparation in the preparation of medicines for treating, preventing, or alleviating tumor-related diseases.

本发明的方法及组合物对于缓解与上述肿瘤相关的一种或者多种症状同样是有效的。The methods and compositions of the present invention are also effective in alleviating one or more symptoms associated with the above-mentioned tumors.

本发明的有益之处在于：本发明的高亲和PD-1蛋白突变体，通过将野生型PD-1的四个氨基酸位点突变：M70I、S87W、A129H、K135M；得到具有更高亲和力的PD-1蛋白突变体，不但可以显著下调T细胞活化和细胞因子分泌，同时还可以降低使用剂量，降低治疗成本。The benefit of the present invention is that the high-affinity PD-1 protein mutant of the present invention is obtained by mutating four amino acid sites of wild-type PD-1: M70I, S87W, A129H, and K135M; The PD-1 protein mutant can not only significantly down-regulate T cell activation and cytokine secretion, but also reduce the dosage and reduce the cost of treatment.

附图说明Description of drawings

图1：pEGFP-N1质粒图谱；酶切位点：Aval，HindIII，ECORI，PstI。Figure 1: Plasmid map of pEGFP-N1; Restriction site: Aval, HindIII, ECORI, PstI.

图2：质粒pEGFP-N1-hPD-1及其突变体(M70I、S87W、A129H、K135M)在HEK-293T细胞膜上的定位。Figure 2: Localization of plasmid pEGFP-N1-hPD-1 and its mutants (M70I, S87W, A129H, K135M) on HEK-293T cell membranes.

图3：流式细胞仪检测hPD-1及其突变体(M70I、S87W、A129H、K135M)与hPD-L1Fc蛋白的结合情况；Figure 3: The binding of hPD-1 and its mutants (M70I, S87W, A129H, K135M) to hPD-L1Fc protein detected by flow cytometry;

图4：hPD-1及其突变体蛋白(K135M)与hPD-L1Fc蛋白亲和力MST实验结果。Figure 4: The results of MST experiments on the affinity between hPD-1 and its mutant protein (K135M) and hPD-L1Fc protein.

具体实施方式Detailed ways

实施例1：pEGFP-N1-hPD-1突变体蛋白重组质粒真核载体的构建。Example 1: Construction of pEGFP-N1-hPD-1 mutant protein recombinant plasmid eukaryotic vector.

将野生型PD-1的四个氨基酸位点突变：M70I、S87W、A129H、K135M，下面详述pEGFP-N1-hPD-1突变体蛋白重组质粒载体的构建。Four amino acid sites of wild-type PD-1 were mutated: M70I, S87W, A129H, K135M, and the construction of pEGFP-N1-hPD-1 mutant protein recombinant plasmid vector was described in detail below.

PCR引物设计：依据hPD-1突变体蛋白的基因序列和密码子表，设计上述四种hPD-1突变体真核载体引物，设计引物如下：PCR primer design: According to the gene sequence and codon table of the hPD-1 mutant protein, design the above four hPD-1 mutant eukaryotic vector primers, and design the primers as follows:

(1)PCR扩增hPD-1突变体蛋白基因反应体系为：30ng PD-1DNA模板：1μL，10mMdNTP Mix：1.5μL，10μM正向引物：0.75μL，10μM反向引物：0.75μL，10×Pfx50buffer：2.5μL，Pfx50DNA polymerase(5U/μL)：1μL，ddH₂O:17.5μL。反应条件为：95℃3min，94℃30s，55℃30s，68℃10min，16个循环后68℃、10min。(1) PCR amplification of hPD-1 mutant protein gene reaction system: 30ng PD-1 DNA template: 1μL, 10mMdNTP Mix: 1.5μL, 10μM forward primer: 0.75μL, 10μM reverse primer: 0.75μL, 10×Pfx50buffer : 2.5 μL, Pfx50 DNA polymerase (5 U/μL): 1 μL, ddH ₂ O: 17.5 μL. The reaction conditions were: 95°C for 3 min, 94°C for 30 s, 55°C for 30 s, 68°C for 10 min, and 68°C for 10 min after 16 cycles.

(2)跑胶和胶回收PCR产物：制备1％琼脂糖凝胶，在PCR产物中加入5μL 6×Loading Buffer，点样并电泳。设置电泳参数：电压：200V，时间：20min。切胶并进行胶回收。(2) Gel running and gel recovery of PCR products: prepare 1% agarose gel, add 5 μL of 6×Loading Buffer to the PCR products, spot and perform electrophoresis. Set electrophoresis parameters: voltage: 200V, time: 20min. Cut glue and carry out glue recovery.

(3)DpnI酶切pEGFP-N1-hPD-1模板，10μL酶切体系：胶回收产物：7.5μL，10×CutSmart buffer：2μL，DpnI-HF酶：0.5μL。将酶切体系混匀，离心，封口放于37℃培养箱中双酶切4个小时，随后置于4℃冰箱中备用。(3) DpnI digestion of pEGFP-N1-hPD-1 template, 10 μL digestion system: Gel recovery product: 7.5 μL, 10×CutSmart buffer: 2 μL, DpnI-HF enzyme: 0.5 μL. Mix the enzyme digestion system, centrifuge, seal and place in a 37°C incubator for double enzyme digestion for 4 hours, and then place it in a 4°C refrigerator for later use.

(4)使用酶切产物转化XL-1Blue感受态细胞，各取100μL分别涂布LB kan平板。次日挑单克隆，摇菌，送金唯智公司测序验证。经验证测序正确，即构建成功了含有hPD-1基因突变体的真核表达载体，分别命名为pEGFP-N1-hPD-1(M70I)、pEGFP-N1-hPD-1(S87W、pEGFP-N1-hPD-1(A129H)、pEGFP-N1-hPD-1(K135M)。(4) Transform XL-1Blue competent cells with the digested products, and take 100 μL of each to spread on LB kan plates. Pick a single clone the next day, shake the bacteria, and send it to Jinweizhi Company for sequencing verification. It was verified that the sequencing was correct, that is, the eukaryotic expression vectors containing hPD-1 gene mutants were successfully constructed, named pEGFP-N1-hPD-1(M70I), pEGFP-N1-hPD-1(S87W, pEGFP-N1- hPD-1(A129H), pEGFP-N1-hPD-1(K135M).

实施例2 pEGFP-N1-hPD-1突变体蛋白重组质粒真核载体的转染Example 2 Transfection of pEGFP-N1-hPD-1 mutant protein recombinant plasmid eukaryotic vector

2.1细胞培养2.1 Cell culture

(1)复苏HEK-293T细胞:打开水浴锅预热到37℃，将新鲜的DMEM培养基放入水浴锅中预热，其次，从-80℃冰箱中取出HEK-293T细胞，快速放在37℃水浴锅中不停振荡，直到冻存的细胞完全融化，置入离心机中，设置转速：1000rpm，离心时间：5min。放于生物安全柜中吸弃上清，向冻存管中加入新鲜的DMEM培养基，并轻轻吹打将细胞重悬起来，最后将细胞悬液转移到细胞培养皿中，补加10mL DMEM培养基，放于37℃，5％CO2恒温培养箱中进行细胞培养。(1) Resuscitation of HEK-293T cells: Turn on the water bath to preheat to 37°C, put fresh DMEM medium into the water bath to preheat, and then take out the HEK-293T cells from the -80°C refrigerator, and place them quickly at 37°C. Keep shaking in a water bath at ℃ until the frozen cells are completely thawed, put them into a centrifuge, set the rotation speed: 1000rpm, and centrifuge time: 5min. Put it in a biosafety cabinet, suck out the supernatant, add fresh DMEM medium to the cryovial, and gently pipette to resuspend the cells. Finally, transfer the cell suspension to a cell culture dish, add 10mL DMEM for culture Cell culture was carried out in a 37°C, 5% CO2 constant temperature incubator.

(2)细胞传代：当细胞长满后，吸弃旧培养基，缓慢加入10mL的pH7.2的PBS溶液，轻轻吸弃PBS溶液，加入1mL 0.25％的胰酶，待1min后，吸弃掉胰酶，加入2mL新鲜的DMEM培养基，终止胰酶消化，用移液枪轻吹培养基，放于显微镜下观察细胞是否成单个细胞，观察后以1：3分皿后，补加10mL DMEM培养基，放入37℃，5％CO₂恒温培养箱中进行细胞培养，在传代期间不时观察细胞状态并逐渐传代扩大培养。(2) Cell subculture: when the cells are full, discard the old medium, slowly add 10 mL of PBS solution with pH 7.2, gently discard the PBS solution, add 1 mL of 0.25% trypsin, wait for 1 min, then discard Remove trypsin, add 2mL of fresh DMEM medium, stop trypsin digestion, gently blow the medium with a pipette gun, put it under a microscope to observe whether the cells are single cells, divide the dishes at a ratio of 1:3 after observation, and then add 10mL DMEM medium was placed in a 37°C, 5% _CO2 constant temperature incubator for cell culture, and the cell state was observed from time to time during the passaging period, and the culture was gradually expanded by passaging.

2.2 CaCl₂瞬时转染法2.2 CaCl ₂ transient transfection method

(1)转染之前18～20小时，铺3～4×106HEK-293T细胞到10cm细胞培养皿中。(1) 18-20 hours before transfection, spread 3-4×106 HEK-293T cells into a 10cm cell culture dish.

(2)显微镜下观察HEK-293T细胞汇合度，至70～80％用于转染，随后转染前2小时细胞重新置换新的含有10％FBS的DMEM培养基。(2) The confluence of HEK-293T cells was observed under a microscope and was used for transfection when it reached 70-80%, and then the cells were replaced with new DMEM medium containing 10% FBS 2 hours before transfection.

(3)配制氯化钙转染试剂：质粒20μg、2.5M CaCl₂ 50μL、ddH₂O，将质粒先补足无菌ddH₂O 450μL，充分混匀后，再将2.5M CaCl₂加入混匀，静置1～2min，此时为质粒-CaCl2的混合物。(3) Prepare calcium chloride transfection reagent: 20 μg of plasmid, 50 μL of 2.5M CaCl ₂ , ddH ₂ O, first supplement the plasmid with 450 μL of sterile ddH ₂ O, mix well, then add 2.5M CaCl ₂ and mix evenly, Let it stand for 1-2min, at this time it is a mixture of plasmid-CaCl2.

(4)用移液器将上述的质粒-CaCl2的混合物加入到500μL 2×HBS中去，边加入边混合均匀，最后，用鼓气泡的方法鼓气大约20次。(4) Add the above-mentioned plasmid-CaCl2 mixture into 500 μL 2×HBS with a pipette, mix well while adding, and finally, use the method of blowing air bubbles for about 20 times.

(5)将步骤(4)混合物静置10～15min，待看到“雾状”液体时，逐滴均匀加入HEK-293T细胞中。(5) Let the mixture in step (4) stand still for 10-15 minutes, and add it dropwise to HEK-293T cells evenly when a "foggy" liquid is seen.

(6)转染6小时后，换新鲜10％FBS的DMEM培养基。(6) After 6 hours of transfection, replace the DMEM medium with fresh 10% FBS.

实施例3细胞膜定位情况判断Example 3 Judgment of Cell Membrane Localization

(1)转染35-40小时后，首先用流式细胞仪器检测转染情况。(1) After 35-40 hours of transfection, first detect the transfection status with a flow cytometer.

(2)弃掉培养基，缓慢加入10mL的pH7.2的PBS溶液，轻轻吸弃PBS溶液，加入1mL0.25％的胰酶，待1min后，吸弃掉胰酶，PBS7.2洗两遍后，收集细胞，制成单细胞悬液，然后按5×105细胞收集到15ml离心管中，随后分装于1.5mL离心管中，3000rpm/min离心5min弃掉流式buffer。(2) Discard the medium, slowly add 10 mL of PBS solution with pH 7.2, gently aspirate and discard the PBS solution, add 1 mL of 0.25% trypsin, wait for 1 min, aspirate and discard the trypsin, wash with PBS7.2 for two After the first pass, the cells were collected to make a single-cell suspension, and then 5×105 cells were collected into 15ml centrifuge tubes, then distributed into 1.5mL centrifuge tubes, centrifuged at 3000rpm/min for 5min, and the flow buffer was discarded.

(3)将anti-hPD-1流式抗体和转染真核载体pEGFP-N1-hPD-1突变体的HEK-293T细胞放于冰上孵育30min，然后用1ml PBS 7.2洗一次，加入200μL Buffer重悬后，最后用流式细胞仪器检测在PD-1在细胞膜上的表达情况。(3) Incubate the anti-hPD-1 flow cytometry antibody and the HEK-293T cells transfected with the eukaryotic vector pEGFP-N1-hPD-1 mutant for 30 minutes on ice, then wash once with 1ml PBS 7.2, add 200μL Buffer After resuspension, the expression of PD-1 on the cell membrane was finally detected by flow cytometry.

实施例4突变体亲和力检测FACSExample 4 Mutant affinity detection FACS

(1)将购买的粉末状真核蛋白Human-PD-L1-Fc蛋白(Catalog Number:10084-H02H)，用PBS7.2完全溶解，制成浓度为0.25mg/mL的蛋白溶液，分装后放于-80℃冰箱中备用。(1) Completely dissolve the purchased powdered eukaryotic protein Human-PD-L1-Fc protein (Catalog Number: 10084-H02H) with PBS7.2 to make a protein solution with a concentration of 0.25mg/mL, and divide into Store in a -80°C freezer for later use.

(2)将转染有pEGFP-N1-hPD-1、pEGFP-N1-hPD-1(M70I)、pEGFP-N1-hPD-1(S87W)、pEGFP-N1-hPD-1(A129H)、pEGFP-N1-hPD-1(K135M)的HEK-293T细胞与不同浓度的真核蛋白Human-PD-L1-Fc在冰上孵育30min，阴性对照组只加荧光二抗(APC anti-human IgG Fc，clone:HP6017)，随后用1mL PBS7.2洗一遍，最后用流式细胞仪检测荧光强度的变化情况。(2) Transfected with pEGFP-N1-hPD-1, pEGFP-N1-hPD-1 (M70I), pEGFP-N1-hPD-1 (S87W), pEGFP-N1-hPD-1 (A129H), pEGFP- N1-hPD-1(K135M) HEK-293T cells were incubated with different concentrations of eukaryotic protein Human-PD-L1-Fc on ice for 30min, and the negative control group was only added with fluorescent secondary antibody (APC anti-human IgG Fc, clone :HP6017), followed by washing with 1mL PBS7.2, and finally using flow cytometry to detect changes in fluorescence intensity.

(3)真核蛋白Human-PD-L1-Fc蛋白的使用浓度分别为：1μM，3μM，10μM，30μM，100μM，300μM。(3) The concentrations of eukaryotic protein Human-PD-L1-Fc protein used are: 1 μM, 3 μM, 10 μM, 30 μM, 100 μM, 300 μM.

实施例5突变体亲和力检测MST结果Example 5 Mutant affinity detection MST result

(1)收集细胞：用胰酶消化平皿中培养的转染成功pEGFP-N1-hPD-1、pEGFP-N1-hPD-1(M70I)、pEGFP-N1-hPD-1(S87W)、pEGFP-N1-hPD-1(A129H)、pEGFP-N1-hPD-1(K135M)的HEK-293T细胞细胞，用含有10％胎牛血清的DMEM重悬细胞，收集到1.5mL的离心管中，800g离心5-10分钟，弃去上清培养基。用PBS7.2洗一遍细胞，再次离心收集细胞。(1) Collect cells: Use trypsin to digest successfully transfected pEGFP-N1-hPD-1, pEGFP-N1-hPD-1(M70I), pEGFP-N1-hPD-1(S87W), pEGFP-N1 - hPD-1(A129H), pEGFP-N1-hPD-1(K135M) HEK-293T cells, resuspended cells in DMEM containing 10% fetal bovine serum, collected in a 1.5mL centrifuge tube, centrifuged at 800g for 5 -10 minutes, discard the supernatant medium. Wash the cells once with PBS7.2, and collect the cells by centrifugation again.

(2)细胞匀浆裂解：离心后每100μL体积细胞沉淀中加入500μL CER试剂，震荡重悬，冰浴2分钟。将冰浴的细胞悬液转移到预冷的1mL小容积的玻璃匀浆器内，在冰上上下手动匀浆20-30次。(2) Cell homogenate lysis: add 500 μL of CER reagent to every 100 μL volume of cell pellet after centrifugation, resuspend by shaking, and ice-bath for 2 minutes. Transfer the ice-bathed cell suspension to a pre-cooled 1mL small-volume glass homogenizer, and manually homogenize it up and down on ice 20-30 times.

(3)粗提物的制备：取约500μL裂解物，转移到新的离心管中，800g，4℃离心5分钟。离心结束，上清是胞膜和胞浆混合物。(3) Preparation of crude extract: Take about 500 μL of lysate, transfer to a new centrifuge tube, centrifuge at 800 g, 4° C. for 5 minutes. After centrifugation, the supernatant is a mixture of membrane and cytoplasm.

(4)胞膜的制备：将上步骤中获得的上清转移到新的离心管中，加入上清十分之一体积的MER与上清液混合，冰浴5分钟，14000rpm，4℃离心30分钟，去上清，沉淀为胞膜组分，其中含有细胞膜和亚细胞器碎片，用50μL含有1％的Triton X-100的1×MST Buffer重悬胞膜，-80℃保存。(4) Preparation of cell membrane: Transfer the supernatant obtained in the previous step to a new centrifuge tube, add 1/10 volume of MER and mix with the supernatant, ice-bath for 5 minutes, centrifuge at 14000rpm, 4°C After 30 minutes, the supernatant was removed, and the cell membrane fraction was precipitated, which contained cell membrane and subcellular organelle fragments. The cell membrane was resuspended in 50 μL of 1×MST Buffer containing 1% Triton X-100, and stored at -80°C.

(5)调整膜蛋白荧光值：先取突变体蛋白每管20μL于200μL的PCR管中，用毛细管将其吸入，然后放到MST机器上检测，当LED Power在5-90％之间，荧光值在300-1500之间较为理想。当检测的荧光值高出这个范围，通常采用稀释膜蛋白的方法，当检测的荧光值低于这个范围时，通常采用增加LED Power来解决。为了控制突变蛋白量的一致性，荧光值都控制800。(5) Adjust the fluorescence value of the membrane protein: First, take 20 μL of each tube of the mutant protein in a 200 μL PCR tube, inhale it with a capillary, and then put it on the MST machine for detection. When the LED Power is between 5-90%, the fluorescence value Ideally between 300-1500. When the detected fluorescence value is higher than this range, the method of diluting the membrane protein is usually used. When the detected fluorescence value is lower than this range, it is usually solved by increasing the LED Power. In order to control the consistency of the mutant protein amount, the fluorescence value was controlled at 800.

(6)将靶蛋白与配体混合：将稀释好带突变体蛋白蛋白放置于冰上，把突变体蛋白稀释16个浓度梯度，将10μL的PD-1突变体蛋白稀释液与10μL hPD-L1-Fc蛋白混合于200μL的管中。(6) Mix the target protein with the ligand: place the diluted mutant protein on ice, dilute the mutant protein in 16 concentration gradients, mix 10 μL of the PD-1 mutant protein dilution with 10 μL hPD-L1 - Fc protein was mixed in a 200 μL tube.

(7)样品检测：将混合后的蛋白用毛细管吸入，放置于MST机器上检测，拟合Kd值。最后用MST分析软件处理数据，绘制拟合曲线，每次实验至少重复3次。(7) Sample detection: suck the mixed protein with a capillary tube, place it on the MST machine for detection, and fit the Kd value. Finally, the data were processed with MST analysis software, and the fitting curve was drawn. Each experiment was repeated at least 3 times.

以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto, any person familiar with the technical field within the technical scope disclosed in the present invention, according to the technical solution of the present invention Any equivalent replacement or change of the inventive concepts thereof shall fall within the protection scope of the present invention.

序列表sequence listing

<110> 郑州大学<110> Zhengzhou University

<120> 一种高亲和PD-1蛋白突变体<120> A high-affinity PD-1 protein mutant

<130> 2010<130> 2010

<141> 2018-05-30<141> 2018-05-30

<160> 8<160> 8

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 170<211> 170

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 1<400> 1

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu GlnMet Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 151 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro TrpLeu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30 20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly AspAsn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45 35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe ValAsn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60 50 55 60

Leu Asn Trp Tyr Arg Ile Ser Pro Ser Asn Gln Thr Asp Lys Leu AlaLeu Asn Trp Tyr Arg Ile Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 8065 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe ArgAla Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95 85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val ArgVal Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110 100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser LeuAla Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125 115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg ValAla Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140 130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser ProThr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu ValArg Pro Ala Gly Gln Phe Gln Thr Leu Val

165 170 165 170

<210> 2<210> 2

<211> 501<211> 501

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 2<400> 2

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatc agccccagca accagacgga caagctggcc 240gagagcttcg tgctaaactg gtaccgcatc agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca a 501aggccagccg gccagttcca a 501

<210> 3<210> 3

<211> 170<211> 170

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 3<400> 3

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu GlnMet Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 151 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro TrpLeu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30 20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly AspAsn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45 35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe ValAsn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60 50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu AlaLeu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 8065 70 75 80

Ala Phe Pro Glu Asp Arg Trp Gln Pro Gly Gln Asp Cys Arg Phe ArgAla Phe Pro Glu Asp Arg Trp Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95 85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val ArgVal Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110 100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser LeuAla Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125 115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg ValAla Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140 130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser ProThr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu ValArg Pro Ala Gly Gln Phe Gln Thr Leu Val

165 170 165 170

<210> 4<210> 4

<211> 501<211> 501

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 4<400> 4

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgctg gcagcccggc caggactgcc gcttccgtgt cacacaactg 300gccttccccg aggaccgctg gcagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca a 501aggccagccg gccagttcca a 501

<210> 5<210> 5

<211> 170<211> 170

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 5<400> 5

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu GlnMet Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 151 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro TrpLeu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30 20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly AspAsn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45 35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe ValAsn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60 50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu AlaLeu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 8065 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe ArgAla Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95 85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val ArgVal Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110 100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser LeuAla Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125 115 120 125

His Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg ValHis Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140 130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser ProThr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu ValArg Pro Ala Gly Gln Phe Gln Thr Leu Val

165 170 165 170

<210> 6<210> 6

<211> 501<211> 501

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 6<400> 6

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctgcatccc aaggcgcaga tcaaagagag cctgcgggca 420tacctctgtg gggccatctc cctgcatccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca a 501aggccagccg gccagttcca a 501

<210> 7<210> 7

<211> 170<211> 170

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 7<400> 7

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu GlnMet Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 151 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro TrpLeu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30 20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly AspAsn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45 35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe ValAsn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60 50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu AlaLeu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 8065 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe ArgAla Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95 85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val ArgVal Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110 100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser LeuAla Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125 115 120 125

Ala Pro Lys Ala Gln Ile Met Glu Ser Leu Arg Ala Glu Leu Arg ValAla Pro Lys Ala Gln Ile Met Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140 130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser ProThr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu ValArg Pro Ala Gly Gln Phe Gln Thr Leu Val

165 170 165 170

<210> 8<210> 8

<211> 501<211> 501

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 8<400> 8

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcatggagag cctgcgggca 420tacctctgtg gggccatctc cctggccccc aaggcgcaga tcatggagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca a 501aggccagccg gccagttcca a 501