CN110759996B - GFP antibody - Google Patents

Detailed Description

The present invention provides a GFP antibody. In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are therefore not to be considered limiting of the invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or may be connected through the use of two elements or the interaction of two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

In the present invention, unless expressly stated or limited otherwise, the first feature "on" or "under" the second feature may be directly contacting the second feature or the first and second features may be indirectly contacting each other through intervening media. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

The embodiment of the invention provides a GFP antibody. The GFP antibody is an anti-GFP antibody capable of specifically binding to GFP. When GFP is used as a tag, a target molecule to which the GFP tag is attached is isolated and purified.

Specifically, the GFP antibody has a relatively simple single chain structure, containing only one heavy chain. More specifically, the molecular weight of the GFP antibody is about 15KD, and the GFP antibody is small in molecular weight and can be suitable for different use scenes.

In some embodiments, the GFP antibody has a variable region on the only heavy chain that specifically binds to the antigen of interest (i.e., GFP).

The single-chain antibody structure can enable the antibody to be well suitable for various different use conditions, the heavy chain and light chain pollution of a typical Y-shaped antibody cannot occur in the downstream application after purification, the single-chain antibody structure can be well applied to a GFP label, the operation flow of a biotechnology experiment is simplified, and the accuracy of the experiment is improved.

The following describes the preparation process of the GFP antibody in detail with reference to specific examples. In this example, the GFP antibody may be a camelid single domain antibody derived from camelids.

11. As an antigen, His-tagged GFP protein recombinantly expressed in E.coli was used, and mixed and emulsified with GERBU-LQ (Gerbu, cat # 3030.0100) to give an immunogen.

12. 2 alpacas were immunized every 2 weeks with the above treated immunogen, 2mg of emulsified immunogen was injected each time around the alpaca neck lymph node. 20ml of blood is drawn from the carotid artery of the alpaca to an anticoagulation tube 3-4 days after the 4 th, 5 th and 6 th immunization respectively, and the alpaca immunization treatment is completed.

13. Blood and leukocyte separation solution extracted from immune alpaca are mixed at a ratio of 1:1, centrifuged at 400g for 30 minutes at room temperature, and the upper layer immune cells of the middle cotton-like layer are aspirated by a pipette.

14. PBS buffer was added to the aspirated immune cells until the volume reaches 10mL, and after centrifugation at 200g for 20 minutes at room temperature to remove the supernatant, PBS buffer was added again at 5mL, and centrifugation at 200g for 20 minutes at room temperature to remove the supernatant.

15. Adding 1mL of Trizol into the cleaned immune cells, uniformly mixing to obtain the peripheral blood lymphocytes of the immune alpaca, and storing the peripheral blood lymphocytes in a refrigerator of-20 ℃ for later use.

16. The peripheral blood lymphocytes obtained by extraction were transferred to a 1.5mL centrifuge tube, and 1/5 volumes of chloroform were added thereto, mixed well, and then left to stand at room temperature for 5 minutes, followed by centrifugation at 12000g for 15 minutes at 4 ℃.

17. The centrifuged supernatant was transferred to a new centrifuge tube and 0.5-1 volume isopropanol was added to the centrifuge tube. After adding isopropanol and standing at room temperature for 10 minutes, the mixture was centrifuged at 12000g at 4 ℃ for 10 minutes.

18. The pellet in the centrifuge tube was washed with 75% ethanol in an equal volume to that of the peripheral blood lymphocytes preserved with Trizol, and after centrifuging at 7500g for 5 minutes at 4 degrees Celsius, the pellet was dissolved in an appropriate amount of RNase-free water to complete the RNA extraction of the peripheral blood lymphocytes.

19. After the completion of the RNA extraction, the extracted RNA of peripheral blood lymphocytes is reverse-transcribed into cDNA using a reverse transcription kit.

20. Specific antibody gene fragments were amplified from reverse transcribed cNDA by PCR.

In this embodiment, the PCR reaction system is: cDNA template 2ul, ALPHVH-C primer 2ul, CALL002 primer 2ul, 10 XTaqBuffer 5ul, dNTP 4ul, Taq (HS)0.25ul, ddH2O to 50 ul.

The reaction conditions of PCR were: 98 ℃ for 3 minutes; 30 seconds at 95 ℃, 30 seconds at 57 ℃, 40 seconds at 68 ℃, 2 seconds for each cycle, and 22 cycles; 68 ℃ for 5 minutes.

21. The resulting PCR amplification product was subjected to 1% agarose gel electrophoresis. The results of agarose gel electrophoresis showed: the PCR amplification product formed two bands of about 1.0kb and about 0.7kb in length. After agarose gel electrophoresis, a 0.7kb band was excised and recovered using a DNA purification kit.

22. And continuing to perform PCR again on the recovered DNA fragments to amplify the specific antibody fragments.

The reaction system of the PCR amplification is as follows: DNA template 2ul, Rhhh-FP primer 2ul, Rhhh-RP primer 2ul, 10 XTaq Buffer 5ul, dNTP 4ul, Taq (HS)0.25ul, ddH2O to 50 ul.

The reaction conditions of PCR were: 98 ℃ for 3 minutes; repeating for 12 cycles at 95 ℃ for 50 seconds, 55 ℃ for 30 seconds and 72 ℃ for 40 seconds; 72 degrees Celsius for 10 minutes.

23. And (4) recovering the DNA of the PCR amplification product by using the DNA purification and recovery kit again.

24. The amplified antibody gene sequence and pADL-10b were digested with BglI.

The enzyme digestion system is as follows: amplifying gene 12ug or pADL-10b vector 3ug, 10 XBglI Buffer 3ul, BglI 4.5ul, and supplementing water to 30 ul. The enzyme digestion reaction conditions are as follows: the reaction was carried out at 37 ℃ for 3-4 hours.

25. And recovering the carrier and the amplified antibody gene after enzyme digestion by using a DNA purification recovery kit after enzyme digestion.

26. The recovered vector and the amplified antibody gene were subjected to ligation reaction (overnight reaction at 4 ℃ C.) to recover a ligation product. The ligation product was dissolved using ultrapure water.

In this embodiment, the ligation reaction system is: pADL-10b vector 200ng, amplified antibody gene 80ng, Takara T4 ligase 2ul, 10 Xligation buffer 5ul, and water until 50 ul.

27. And (3) placing the electric rotating cup on ice for precooling, adding 1ul of connecting product after the SS320 competent cells are melted, transferring the mixed competent cells and connecting product into the electric rotating cup which is precooled, and performing electric shock transformation by using a Bacteria transformation program preset by an electric rotating instrument.

28. Immediately after the electroporation, 1mL of SOC medium was added to the cuvette, and the cells were thawed at 37 ℃ for 60 minutes and plated on LB plates containing tetracycline and ampicillin resistance for overnight growth.

29. The cells on the plate were washed with LB medium and a coating rod and scraped, and 20% glycerol was added and stored at-80 ℃ for further use.

30. Cell counting was performed, and about 10^9 transformed cells were transferred to 100mL of 2XYT medium to which tetracycline and ampicillin were previously added, and cultured at 37 ℃ and 220rpm until OD600 reached 0.5.

31. After adding helper phage at a ratio of 20:1 bacterial cell number, incubation was continued at 37 ℃ for 30 minutes. Kanamycin and 0.2mM IPTG were added to the final concentration, and the mixture was cultured overnight at 30 ℃.

32. After 5 minutes of low temperature centrifugation at 13000rpm at 4 degrees Celsius for overnight cultured cells, the supernatant was transferred to a new centrifuge tube and 1/4 volumes of pre-cooled 5X PEG8000/NaCl were added and incubated on ice for 30 minutes.

33. After incubation, the supernatant was removed by centrifugation at 13000rpm for 10 minutes at 4 ℃ and 1mL of PBS buffer was added to dissolve the pellet.

34. The phage library was prepared by adding 250ul of 5X PEG8000/NaCl and incubating on ice for 10 min, 4 ℃ and centrifuging at 16000g for 15 min, then removing the supernatant and dissolving the pellet in 1ml PBS.

35. To each immune tube, 100ug of GFP antibody and 2mL of PBS were added and incubated overnight at 4 degrees celsius to coat the immune tubes.

36. Phage 500ul after amplification and purification of phage library was added to 1mL of 3% BSA and incubated for 2h at room temperature with rotation. Meanwhile, 2-3mL of 3% BSA was added to the coated immune tubes, and the tubes were incubated for 2h at room temperature with rotation.

37. The blocked immune tubes from step 35 were washed 3 times for 5 minutes each with PBS containing 0.01% tween. Then, the blocked phage library was added to the blocked immune tube, PBS was added to 2-3mL, and the tube was incubated at room temperature for 1h with rotation.

38. The antigen and phage incubated immune tube containing 0.01% Tween PBS wash 20 times, each time for 5 minutes.

39. Add 1mL of 100mM Trimethylmeme to the tube, incubate for 10 minutes at room temperature, add 1M Tris-HCl to neutralize the Trimethylmeme, and transfer the final 1.5mL of eluted phage to a fresh centrifuge tube.

40. The eluted phage is subjected to the steps of amplifying and purifying the phage library again. After amplification, the screening process is repeated for 2 times, the amount of the antibody coating the immune tube is gradually reduced by half, and finally the eluted phage after 3 times of screening is obtained.

In this example, two different methods, ELISA and Native SDS-PAGE gel, can be used to perform the identification at different molecular levels.

Example 1(ELISA identification):

firstly, diluting the screened phage by 10^6 times, adding 100ul of the diluted phage into SS320 bacterial liquid with OD600 of 0.5, culturing at 37 ℃ for 30 minutes, coating on a 2XYT culture plate containing tetracycline and ampicillin, and culturing at 37 ℃ overnight for the next day to obtain a monoclonal colony.

Then, 96 monoclonal bacteria were selected and dropped on a 96-well cell culture plate containing 2XYT culture solution of tetracycline and ampicillin, and after 3 to 4 hours of culture at 37 degrees Celsius, kanamycin and 20:1 helper phage were added to the culture well, and overnight culture was carried out at 30 degrees, and the cell broth after overnight culture was centrifuged the next day to obtain a supernatant.

Then, the supernatant obtained in the previous step was added to the 96-well ELISA plate coated with GFP antigen overnight and blocked with 3% BSA, and incubated at room temperature for 1 h. After incubation, the wells were washed 3 times with PBS containing 0.05% Tween, and absorbance was read at a wavelength of 450 after TMB development using anti-fd-Bacteriophage antibody (SIGMA, cat # B7786-.2ML) as a primary antibody and anti-Rabbit antibody coupled to HRP (CWBIO, cat # CW0103S) as a secondary antibody.

And finally, selecting the SS320 colony with the highest light absorption value reading as a sample to perform DNA sequencing, and identifying to obtain the gene sequence of the GFP antibody.

Example 2(Native SDS-PAGE gel identification):

first, SS320 colonies from example 1 above were picked, inoculated into 3mL of 2XYT medium containing tetracycline and ampicillin, and incubated at 37 ℃ until OD600 reached 0.6.

Then, after further culturing for 3 to 6 hours by adding 0.2uM IPTG, the pellet was collected by centrifugation and 100. mu.l of lysate was added, and after standing at room temperature for 10 minutes, the supernatant was collected by centrifugation. 18ul of the supernatant obtained from the preparation was incubated with 1ul of GFP protein for 20 minutes.

It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.

Sequence listing

<110> Shenzhen health life science and technology Limited

<120> GFP antibody

<141> 2018-07-27

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