A CD45 recombinant antibody

Abstract

The application provides a CD45 recombinant antibody, which belongs to the technical field of recombinant antibodies, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ_1, the amino acid sequence of the light chain variable region is shown in SEQ_2, the nucleotide sequence of the coding gene of the heavy chain variable region is shown in SEQ_3, and the nucleotide sequence of the coding gene of the light chain variable region is shown in SEQ_4; the recombinant antibody is obtained by connecting the light chain and the heavy chain to expression vectors respectively and then co-transfecting host cells; the application can produce more stable antibodies and overcome the problems caused by non-specificity (unreliability) and batch difference of traditional antibodies.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of recombinant antibodies, and particularly relates to a CD45 recombinant antibody. BACKGROUND

[0002] Antibodies are the main force in the field of biological science, but its reliability repeatedly puts researchers in a difficult position. Nowadays, the most common problems encountered by researchers mainly include that the purchased antibody for detecting protein X preferentially binds to protein Y, and even can not bind to X at all; poor repeatability, the results cannot be repeated when a new antibody is used to repeat a previous experiment. A good project is thus shelved, and the unreliability of antibodies is very alarming. Different results obtained from the same antibody in several parallel experiments will have disastrous consequences, and the cause of this result may be related to the production process of the antibody. Insufficient specificity, sensitivity and batch difference lead to false scientific findings and a large waste of scientific effort; the unreliability of antibodies causes huge losses in the fields of cancer, metabolism, aging, immunology and cell signal transduction, and any research on complex biological molecules; the time and resource waste caused by antibodies is very huge.

[0003] Traditional antibody preparation is to obtain a single antibody and a polyclonal antibody by immunizing an animal, and the polyclonal antibody is prepared by collecting the blood of the animal after the animal is stimulated by the target antigen and immunized, as long as the animal is alive, the polyclonal antibody can be provided; the monoclonal antibody is obtained by fusing the B cells that can recognize and respond to the target protein with myeloma cells after the host animal is immunized by the target protein, so that the cells can be permanently cultured to continuously produce the target antibody.

[0004] In contrast, recombinant antibodies are different from traditional monoclonal antibodies, because their preparation only needs to immunize animals in the early stage, and even does not need to involve animals. The gene sequence producing the antibody is detected, the sequence is set by itself, and whether the produced protein meets the target protein is detected; and then the gene is inserted into a suitable cell strain to produce the antibody. Because the antibody is determined, even if the original cell strain dies or mutates, the required cell strain can be produced by gene insertion.

[0005] More and more scientists believe that monoclonal antibodies and polyclonal antibodies will eventually be completely replaced by "more structurally clear" recombinant antibodies. Many proteins cannot be recognized by existing reagents because polyclonal antibodies with unclear structures are used, and the use of some gene-recognizable or stored reagents can overcome this. Although polyclonal antibodies have wide availability and are currently the cheapest antibodies for research and development, they are suitable for preparing some less studied antibodies. However, with the continuous clarification of the structure and function of target proteins and the demand for clinical transformation, recombinant cloned antibodies will also have great potential. SUMMARY

[0006] In view of the above, the present application provides a CD45 recombinant antibody, which is more stable by inserting a desired gene of the antibody into a cell strain, thereby overcoming problems of conventional antibodies due to unreliability and batch difference.

[0007] To solve the above technical problem, the present application provides a CD45 recombinant antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region, characterized in that the amino acid sequence of the antibody heavy chain variable region is shown in SEQ_1, and the amino acid sequence of the light chain variable region is shown in SEQ_2.

[0008] QVKLQQSGAELARPGGSVKLSCKASGYSFTAYWMQWLRQSPGQGLEWIGVIYPGDGDARYTQKFQGKATLTADKSSSTAYMQLSSLASEDSAVYYCARWFHHDYVMDYWGQGTTVTVSS -SEQ_1;

[0009] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHTDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQNTHVPPTFGGGSRLEIK -SEQ_2.

[0010] The CD45 recombinant antibody comprises a heavy chain variable region and a light chain variable region, the nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ_3, and the nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ_4.

[0011] caggtgaagctgcagcagtctggggctgagctggcaagacctg

[0012] ggggttcagtgaagttgtcctgcaaggcttctggctacagctttactgcctactggatgc

[0013] agtggttaagacagagccctggacagggtctggagtggattggagttatttatcctggag

[0014] atggtgatgctaggtatactcagaagttccagggcaaggccacattgactgcagataaat

[0015] cctccagcac agcctacatg caactcagc agcttggca ctctgagga ctctgcggtc tatt

[0016] actgtgcaag atggttcca ccatgacta tgttatgga ctactgggg ccaagggac cacgg

[0017] tcaccgtctcctcaa -SEQ_3.

[0018] gatgttgtga tgacccaaa ctccactctc ctgcctgtcagtcttggaga tcaagcctcc atctcctgca gatctagtc agagccttgt acacactga tggaaacac ctatttaca ttggtacct gcagaagcc aggccagtc tccaaagctc ctgatctac aaagtttcc aaccgattt tctggggtc ccagacagg ttcagtggcagtggatcag ggacagatt tcacactca agatcagca gagtggagg ctgaggatc tgggacttt atttctgct ctcagaata cacatgttc ctccgacgt tcggtggag gctccaggt ggaaatcaa ac -SEQ_4.

[0019] Primer for amplifying the gene encoding the heavy chain variable region of the antibody, the nucleotide sequence of which is as follows:

[0020] 33331 20201016 IgVMu VH1BACK (FR1) - IgVMu - upstream:

[0021] AGGTSMARCTGCAGSAGTCWGG -SEQ_5;

[0022] 33336 20201016 IgVMu HindIII / MH (γ) - CONST - IgVMu downstream:

[0023] GGAAGCTTAYCTCCACACACAGGRRCCAGTGGATAGAC -SEQ_6.

[0024] Primer for amplifying the gene encoding the light chain variable region of the antibody, the nucleotide sequence of which is as follows:

[0025] 33337 20201016 IgVMuEcoRI / FR1-ML(k)-IgVMu upstream:

[0026] GGGAATTCGAYATTGTGMTRACMCARKMTCAA -SEQ_7;

[0027] 33338 20201016 IgVMuHindIII / ML(k)-CONST-IgVMu downstream:

[0028] GGAAGCTTACTGGATGGTGGGAAGATGGA -SEQ_8.

[0029] A recombinant plasmid containing a nucleotide sequence of a coding gene of a heavy chain variable region as shown in SEQ_3 and a nucleotide sequence of a coding gene of a light chain variable region as shown in SEQ_4.

[0030] A primer for secondary amplification of CD45 recombinant antibody, the nucleotide sequence of the primer is as follows:

[0031] Mouse-EcoRI / VH-F1(cd45-C17-2nd) upstream:

[0032] CGGAATTCcaggtgaagctgcagcagtct -SEQ_9;

[0033] Mouse-Nhel / VH-R1(cd45-C17-2nd) downstream:

[0034] CTAGCTAGC TGAGGAGACGGTGACCGTGGT -SEQ_10;

[0035] Mouse-EcoRI / VL-F1(cd45-C17-2nd) upstream:

[0036] CGGAATTCgatgttgtgatgacccaaactc -SEQ_11;

[0037] Mouse-Xhol / VL-R1(cd45-C17-2nd) downstream:

[0038] CCGCTCGAG TTTGATTTCCAGCCTGGAGCCTCCA -SEQ_12.

[0039] The beneficial effects of the above technical solutions of the present application are as follows:

[0040] Compared with the traditional monoclonal antibody and polyclonal antibody, the gene sequence of the recombinant antibody is determined, and the related personnel can perform pre-detection and post-detection when using the recombinant antibody, so as to ensure the stability of the recombinant antibody, and the polyclonal antibody is not suitable for clinical transformation because the structure is not clear and the structure and function of the target protein are continuously clear, so the recombinant antibody is more complete and specific in research and analysis, and is easy to analyze. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The recombinant antibody expression flowchart of the application;

[0042] Figure 2 The total RNA (A) and cDNA internal standard amplification agarose gel electrophoresis (B) of the mouse spleen cell and SP20 fusion hybridoma cell of the application;

[0043] Figure 3 The mouse monoclonal antibody variable region gene amplification of the application;

[0044] Figure 4 The mouse monoclonal antibody variable region gene T clone amplification diagram of the application;

[0045] Figure 5 The CD45 recombinant antibody light chain variable region gene sequencing result of the application;

[0046] Figure 6 The CD45 recombinant antibody light chain variable region sequence analysis result of the application;

[0047] Figure 7 The CD45 recombinant antibody heavy chain variable region gene sequencing result of the application;

[0048] Figure 8 The CD45 recombinant antibody heavy chain variable region sequence analysis result of the application;

[0049] Figure 9 The SDS-PAGE detection diagram of the antibody expressed and purified in the application;

[0050] Figure 10 The WB detection CD45 recombinant antibody and the binding activity diagram of the recombinant human CD45 antigen of the application;

[0051] Figure 11 The ELISA detection CD45 recombinant antibody and the binding activity diagram of the recombinant human CD45 antigen of the application.

[0052] Figure 12 The FCM detection CD45 recombinant antibody and the binding activity diagram of the PBMC of the application;

[0053] Figure 13 This is a diagram illustrating the binding activity of the recombinant CD45 antibody to CD45 on the Jurakat cell membrane, as detected by immunofluorescence in this invention. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will be described in conjunction with the accompanying drawings of the embodiments of the present invention. Figures 1-13 The technical solutions of the embodiments of the present invention will be clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention. In the following embodiments, VH represents the heavy chain variable region, and VL represents the light chain variable region.

[0055] Example 1

[0056] This embodiment provides a complete preparation process for CD45 recombinant antibody, and the preparation method is as follows:

[0057] First, the variable region gene of the mouse monoclonal antibody was extracted.

[0058] 1. Results of RNA extraction and reverse transcription from hybridoma cells:

[0059] After obtaining hybridoma cells fused with mouse spleen cells and SP20, RNA was extracted from the hybridoma cells using the Trizol method, such as... Figure 2 As shown, clear 28S and 18S bands were observed by agarose gel electrophoresis, indicating good RNA integrity. The RNA concentration and purity measurement results were D(260 nm) / D(280 nm) = 1.85, which meets the requirements of this experiment.

[0060] cDNA was synthesized by reverse transcription using RNA as a template. Using cDNA as a template and mouse internal reference gene β-actin as a primer, PCR amplification was performed, and a target band of 380 bp in length was amplified, indicating that the reverse transcribed cDNA can be used for subsequent experiments.

[0061] 2. PCR amplification of the variable region of mouse monoclonal antibody

[0062] Primer sequences:

[0063] Using multiple sequence alignment and degenerate primer design algorithms, primers capable of amplifying the variable region gene of mouse monoclonal antibodies were designed in the relatively constant region of the variable region and the leader peptide of the mouse monoclonal antibody. The sequences are as follows:

[0064] Mouse heavy chain variable region primer sequence

[0065] 33331 20201016 IgVMu VH1BACK(FR1)-IgVMu-Upstream:

[0066] AGGTSMARCTGCAGSAGTCWGG - SEQ 5;

[0067] 33336 20201016 IgVMu HindIII / MH(γ)-CONST-IgVMu downstream:

[0068] GGAAGCTTAYCTCCACACACAGGRRCCAGTGGATAGAC - SEQ 6.

[0069] Mouse light chain variable region primer sequence

[0070] 33337 20201016 IgVMuEcoRI / FR1-ML(k)-IgVMu upstream:

[0071] GGGAATTCGAYATTGTGMTRACMCARKMTCAA - SEQ 7;

[0072] 33338 20201016 IgVMu HindIII / ML(k)-CONST-IgVMu downstream:

[0073] GGAAGCTTACTGGATGGTGGGAAGATGGA - SEQ 8.

[0074] The V region gene of McAb was amplified by Taq DNA enzyme with cDNA as template. The VH upstream primer and VH downstream primer were mixed in a certain proportion to amplify the complete set of VH genes; the VL upstream primer and VL downstream primer were mixed in a certain proportion to amplify the complete set of VL genes. As shown in the agarose gel electrophoresis results of the amplification products, the length of VH gene fragment was about 350-400 bp, and the length of VL gene fragment was about 350 bp, which was consistent with the length of the target fragment; Figure 3

[0075] 3. Purification of V region gene and T-vector ligation:

[0076] The PCR product was purified by gel recovery kit, and the VH gene and VL gene were respectively ligated with pGEM-T vector and transformed into E. coli. As shown in the blue-white screening, Figure 4 the white colonies were selected and identified by PCR. Each of the ligands selected 12 single colonies, and the universal primer was used for colony PCR. The size of the target fragment was about 500 bp, as shown in the agarose gel electrophoresis results of the amplification products, and 6 positive clones with bright bands were sent to the sequencing company for sequencing; Figure 4

[0077] 4. Sequencing results of variable region sequence of CD45 recombinant antibody: ​​

[0078] 4.1 The sequencing results of the variable region of the light chain of the CD45 recombinant antibody are as follows:

[0079] Sequencing results of the CD45 recombinant antibody light chain variable region were obtained, such as... Figure 5 As shown. Homology comparisons of light chain genes were performed using the IMGT / QUEST online analysis software. The results are shown in [link to analysis]. Figure 6 The functional light chain variable region is 337 bases long, and the domain starts from the first base, encoding 112 amino acids. All functional light chains belong to the Musmus IGKV1-110*01F family, with a V region matching rate of 97.96% and a J region matching rate of 94.29%.

[0080] The valid sequences of the light chain genes are as follows:

[0081] gatgttgtgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcctgcagatctagtcagagccttgtacacactgatggaaacacctatttacattggtacctgcagaagccaggccagtctccaaagctcctgatctacaaaagtt tccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggactttatttctgctctcagaatacacatgttcctccgacgttcggtggaggctccaggctggaaatcaaac -SEQ_4.

[0082] 4.2 The sequencing results of the heavy chain variable region of the CD45 recombinant antibody are as follows:

[0083] The sequencing results of the heavy chain variable region of the CD45 recombinant antibody were obtained, such as... Figure 7 As shown. Figures 7-8 As shown, homology comparisons of heavy chain variable region genes were performed using the IMGT / QUEST online analysis software. The analysis results are shown in [link to analysis]. Figure 8 The functional heavy chain variable region is 358 bases long, and the domain starts from the first base and encodes 119 amino acids. All functional heavy chains of this monoclonal antibody belong to the Musmus IGHV1-87*01F family, with a V region matching rate of 93.06% and a J region matching rate of 87.04%.

[0084] The valid sequences are as follows:

[0085] caggtgaagctgcagcagtctggggctgagctggcaagacctgggggttcagtgaagttgtcctgcaaggcttctggctacagctttactgcctactggatgcagtggttaagacagagccctggacagggtctggagtggattggagttatttatcctggagatggtgatgctaggtatactcagaagttccagggcaaggccacattgactgcagataaatcctccagcacagcctacatgcaactcagcagcttggcatctgaggactctgcggtctattactgtgcaagatggttccaccatgactatgttatggactactggggccaagggaccacggtcaccgtctcctcaa -SEQ_3;

[0086] Reverse complement:

[0087] ttgaggagacggtgaccgtggtcccttggccccagtagtccataacatagtcatggtggaaccatcttgcacagtaatagaccgcagagtcctcagatgccaagctgctgagttgcatgtaggctgtgctggaggatttatctgcagtcaatgtggccttgccctggaacttctgagtatacctagcatcaccatctccaggataaataactccaatccactccagaccctgtccagggctctgtcttaaccactgcatccagtaggcagtaaagctgtagccagaagccttgcaggacaacttcactgaacccccaggtcttgccagctcagccccagactgctgcagcttcacctg -SEQ_13.

[0088] II. Full length antibody construction and purification of CD45 recombinant antibodies in eukaryotic expression:

[0089] 1. According to the above-mentioned sequencing confirmation correct and functional heavy chain and light chain variable region gene, combined with the restriction sites of expression vector and reading frame, the secondary amplification primer of antibody amplification is designed, and the corresponding T clone is used as a template for secondary PCR. The PCR product is treated with restriction enzyme, and then connected into the modified pFUSEss_CHIg_mouseG2B and pFUSE2ss_CLIg_mouseK respectively, the plasmid recombination is completed, and the recombinant plasmid is transformed into DH5a, and the positive clone is selected and sent to the company for sequencing;

[0090] The secondary primer design sequence is as follows:

[0091] Mouse-EcoRI / VH-F1 (cd45-C17-2nd) upstream:

[0092] CGGAATTCcaggtgaagctgcagcagtct -SEQ_9;

[0093] Mouse-Nhel / VH-R1 (cd45-C17-2nd) downstream:

[0094] CTAGCTAGC TGAGGAGACGGTGACCGTGGT -SEQ_10;

[0095] Mouse-EcoRI / VL-F1 (cd45-C17-2nd) upstream:

[0096] CGGAATTCgatgttgtgatgacccaaactc -SEQ_11;

[0097] Mouse-Xhol / VL-R1 (cd45-C17-2nd) downstream:

[0098] CCGCTCGAG TTTGATTTCCAGCCTGGAGCCTCCA -SEQ_12;

[0099] 2. The correct plasmid corresponding to the bacterial liquid is expanded and cultured, and the plasmid is extracted. The plasmid carrying the heavy chain and light chain genes is co-transfected into mammalian cells 293F cells or CHO-S cells at a certain ratio, and the culture supernatant of the cells is collected after 4-5 days for protein L HP affinity chromatography purification. The detection results of the purified antibody SDS-PAGE electrophoresis analysis are shown in Figure 9 .

[0100] Example Two

[0101] In this embodiment, the activity of the recombinant antibody is detected by WB

[0102] WB: The sample to be tested recombinant human CD45, incubated with CD45 recombinant antibody at a concentration of 1 ug / mL, and the secondary antibody is HRP-goat anti-mouse IgG (0.5 ug / mL), the results are as follows Figure 10 It is shown that the CD45 recombinant antibody can well recognize the eukaryotic expression of recombinant protein (50 kd);

[0103] Example Three

[0104] This example detects the activity of the recombinant antibody by performing ELISA titer determination

[0105] ELISA titer determination: plate with recombinant human CD45 (293F expression) antigen (1 ug / m1), detect by indirect ELISA method, add CD45 recombinant antibody with gradient dilution (1:100 to 1:512w dilution), the results are as follows Figure 11 It is shown that the CD45 recombinant antibody can specifically bind to the recombinant human CD45 protein, has a good dose-effect relationship, and can still recognize the human CD45 antigen when the antibody is diluted 512w times, predicting that the titer of the recombinant antibody is 512w;

[0106] Example Four

[0107] This example detects the activity of the recombinant antibody by performing flow cytometry

[0108] Flow cytometry detects the binding properties of CD45 recombinant antibody to human PBMC: take fresh human blood, ACK lysis red blood cells and perform blocking treatment, then resuspend the cells to 10 6 ug / 100 μL, incubate with primary CD45 recombinant antibody (10 ug / mL) and secondary APC-goat anti-mouse IgG (10 ug / mL) for flow cytometry detection, and obtain results as follows Figure 12 Conclusion: The CD45 recombinant antibody has good binding activity to natural samples and can be well used for flow cytometry detection.

[0109] Example Five

[0110] Immunofluorescence staining of fixed jurkat with CD45 recombinant antibody.

[0111] Take the cultured Jurkat cells to the EP tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 4% paraformaldehyde and blow evenly, and fix. After fixation, centrifuge, wash with PBS, and repeat three times. Add an appropriate amount of deionized water in the EP tube to prepare a cell suspension, drop it on a polylysine-treated glass slide, air dry, and prepare a Jurkat cell slide. Permeabilize with 0.5% Triton X-100 or not, wash with PBS three times, block with goat serum, discard the blocking solution, add CD45 recombinant antibody (30 ug / mL) and incubate overnight, wash with PBST three times, add secondary antibody Alexa Fluor® 488-goat anti-mouse IgG (2 ug / mL), wash with PBST three times, add DAPI to stain the nucleus, and observe under a fluorescence microscope to obtain results as shown in Figure 13 Conclusion: The CD45 recombinant antibody has good binding activity with the natural sample and can be well used for immunofluorescence detection.

[0112] The amino acid sequence of the heavy chain variable region of the CD45 recombinant antibody is translated as follows:

[0113] QVKLQQSGAELARPGGSVKLSCKASGYSFTAYWMQWLRQSPGQGLEWIGVIYPGDGDARYTQKFQGKATLTADKSSSTAYMQLSSLASEDSAVYYCARWFHHDYVMDYWGQGTTVTVSS -SEQ_1;

[0114] The amino acid sequence of the light chain variable region of the CD45 recombinant antibody is translated as follows:

[0115] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHTDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQNTHVPPTFGGGSRLEIK -SEQ_2.

Claims

1. A CD45 recombinant antibody comprising an antibody heavy chain variable region and an antibody light chain variable region, characterized in that: The amino acid sequence of the antibody heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.

2.

2. The gene encoding CD45 recombinant antibody as claimed in claim 1, wherein, The nucleotide sequence of the coding gene of the heavy chain variable region is shown as SEQ ID NO. 3, and the nucleotide sequence of the coding gene of the light chain variable region is shown as SEQ ID NO.

4.

3. A recombinant plasmid, characterized in that, The coding gene of claim 2.

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