CN117004700A - Method for high-throughput sequencing of monoclonal antibody variable region genes, composition and kit used by method - Google Patents
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The invention discloses a method for high-throughput sequencing of a monoclonal antibody variable region gene in the fields of determination and detection of nucleic acid, and a composition and a kit used by the method. The technical problem to be solved by the invention is how to improve the sequencing efficiency of the antibody variable region. The method for high-throughput sequencing of the variable region genes of the monoclonal antibody comprises the following steps: 1) Obtaining cDNA of hybridoma cells secreting monoclonal antibodies; 2) Performing PCR amplification by using cDNA as a template and using a primer composition to obtain a PCR product; the primer composition consists of a heavy chain primer (comprising a heavy chain upstream primer and a heavy chain downstream primer) and a light chain primer (comprising a light chain upstream primer and a light chain downstream primer); the 5' end of the heavy chain downstream primer and the light chain downstream primer both contain barcode sequences; 3) Mixing PCR products for sequencing; and (3) carrying out data analysis on the sequencing result to obtain the sequence of the monoclonal antibody variable region gene.
Description
Technical Field
The invention relates to a method for high-throughput sequencing of a monoclonal antibody variable region gene in the fields of determination and detection of nucleic acid, and a composition and a kit used by the method.
Background
Hybridoma cell technology is the most widely used and mature platform for antibody production. The hybridoma cells are fused cells of mouse myeloma cells and B lymphocytes. It has the characteristics of infinite proliferation of myeloma cells and the capability of lymphocyte to secrete specific antibodies. Despite the immortalized nature of hybridoma cells, there is still a risk of decreased or lost antibody secretion, and contamination during cell culture or improper storage of cells can also lead to loss of antibodies. If the antibody variable region sequence is obtained through sequencing, constructing the antibody variable region sequence into an expression vector, and obtaining the corresponding antibody through recombinant expression. Thus, it is very important to obtain the antibody variable region sequence by sequencing, which enables the antibody to be preserved in the form of a base sequence.
Existing antibody variable region sequencing is the application of a generation of sequencing technology. One generation of sequencing is the DNA end termination method sequencing technology invented by Sanger et al in the 70 s of the 20 th century by an extension reaction under the action of DNA polymerase, template, radioisotope-labeled primer, dNTP and ddNTP. The presence of ddNTPs resulted in the formation of DNA extension fragments of varying lengths, and the resulting products of the 4 reactions were then separated by slab gel electrophoresis using 4 electrical lanes, allowing the corresponding DNA sequences to be read out sequentially. The Sanger sequencing technology of the first generation has the advantages of long sequencing reading length, short sequencing time, high accuracy and the like.
The antibody sequencing process first extracts hybridoma cell RNA and reverse transcribes it into cDNA. The variable region sequence of the antibody was amplified using specific primers using cDNA as a template, and the variable region sequence was subjected to first-generation sequencing. Although the first generation sequencing technology can meet the requirement of antibody sequencing, each sequencing reaction can only complete the sequencing of one antibody light chain or heavy chain variable region, and if the sequencing of an amplified product is unsuccessful (such as the sequencing result shows a sleeve peak, the signal is weak, and the sequencing of the product fails), TA cloning is also needed. Therefore, the existing method for sequencing is high in sequencing cost, low in flux, time-consuming and labor-consuming, and cannot meet the requirement of high-flux sequencing of the antibody variable region.
With the development of the second generation sequencing technology, the sequencing project greatly reduces the sequencing cost, simultaneously greatly improves the sequencing speed, and maintains high accuracy, and the second generation sequencing is lower than the first generation sequencing in terms of the sequence reading length, but can completely meet the requirement of antibody sequencing. Based on innovations in sequencing technology and the demand for antibody sequencing throughput, development of a second generation sequencing-based antibody high throughput sequencing method is urgently needed.
Disclosure of Invention
The invention aims to solve the technical problems of improving the sequencing efficiency of antibody variable region genes and reducing the sequencing cost.
In order to solve the technical problems, the invention provides a method for high-throughput sequencing of a monoclonal antibody variable region gene.
The invention provides a method for high-throughput sequencing of a monoclonal antibody variable region gene, which comprises the following steps:
1) Obtaining cDNA of N kinds of hybridoma cells, wherein each hybridoma cell in the N kinds of hybridoma cells secretes a monoclonal antibody, and N is a natural number greater than or equal to 2;
2) Using cDNA of each hybridoma cell as a template, and respectively carrying out PCR amplification on a heavy chain variable region gene and a light chain variable region gene of the monoclonal antibody secreted by each hybridoma cell by using a heavy chain variable region gene primer composition and a light chain variable region gene primer composition to obtain a heavy chain variable region gene PCR product of the monoclonal antibody secreted by each hybridoma cell and a light chain variable region gene PCR product of the monoclonal antibody secreted by each hybridoma cell; mixing and sequencing heavy chain variable region gene PCR products of the monoclonal antibodies secreted by the N hybridoma cells to obtain sequencing results of the heavy chain variable region gene PCR products of the monoclonal antibodies secreted by the N hybridoma cells; mixing and sequencing the light chain variable region gene PCR products of the monoclonal antibodies secreted by the N hybridoma cells to obtain the sequencing result of the light chain variable region gene PCR products of the monoclonal antibodies secreted by the N hybridoma cells;
The heavy chain variable region gene primer composition consists of a heavy chain variable region gene upstream primer and a heavy chain variable region gene downstream index primer, wherein the heavy chain variable region gene downstream index primer consists of N primers, the 5' end of each primer contains a Barcode sequence (sequence specific tag), the other heavy chain variable region gene specific sections, and the Barcode sequence of each heavy chain variable region gene downstream index primer is different;
the light chain variable region gene primer composition consists of a light chain variable region gene upstream primer and a light chain variable region gene downstream index primer, wherein the light chain variable region gene downstream index primer consists of N primers, the 5' end of each primer contains a Barcode sequence (sequence specific tag), the rest is a light chain variable region gene specific section, and the Barcode sequence of each light chain variable region gene downstream index primer is different;
3) And 2) carrying out data analysis on the sequencing result of the heavy chain variable region gene PCR products of the monoclonal antibodies secreted by the N hybridoma cells obtained in the 2) and the sequencing result of the light chain variable region gene PCR products of the monoclonal antibodies secreted by the N hybridoma cells, so as to respectively obtain the sequences of the monoclonal antibody variable region genes secreted by each hybridoma cell.
In the method, the Barcode sequence is single-stranded DNA for distinguishing heavy chains or light chains of antibodies of different hybridoma cells. The Barcode sequence may be a single-stranded DNA consisting of 8 nucleotides.
The Barcode sequence may specifically be as follows:
b1, 5'-TCGTCATG-3'; b2, 5'-GTCAGTCC-3'; b3, 5'-CGATTACG-3'; b4, 5'-TCTACGAG-3'; b5, 5'-TAACGTGC-3'; b6, 5'-CGAAGGTT-3'; b7, 5'-CACGCATT-3'; b8, 5'-TACCAGGT-3'; b9, 5'-TTACCGAG-3'; b10, 5 '-AGTCACT-3'; b11, 5'-TCGCATTC-3'; b12, 5'-CATGGTAC-3'; b13, 5 '-CACGGGACT-3'; b14, 5'-CTGTAGCA-3'; b15, 5'-AGAGCTTG-3'; b16, 5'-AGCATAGC-3'; b17, 5 '-CCTAACAT-3'; b18, 5'-GTCGTTGA-3'; b19, 5'-TGCGTATC-3'; b20, 5 '-AGCTACC-3'; b21, 5'-GAACGGTA-3'; b22, 5'-GGATTGTC-3'; b23, 5'-TACCGTCT-3'; b24, 5'-CGAGTATC-3'; b25, 5'-TGTACGCT-3'; b26, 5'-TCCTCTGA-3'; b27, 5'-AGCGTATC-3'; b28, 5 '-TCACC-3'; b29, 5'-GACAGTAC-3'; b30, 5 '-GTCCTTC-3'; b31, 5'-GGAGCAAT-3'; b32, 5'-AGGAACAG-3'; b33, 5'-CAGTGCTA-3'; b34, 5 '-CTAGCTAGTAG-3'; b35, 5'-TGCAAGCA-3'; b36, 5'-CGCATCTA-3'; b37, 5'-CGTATCTG-3'; b38, 5'-GTAGACCT-3'; b39, 5'-CGCCATTA-3'; b40, 5'-ATGACACC-3'; b41, 5'-CAAGTTGA-3'; b42, 5'-TGCTGAAG-3'; b43, 5'-ATGACCGT-3'; b44, 5'-TGGCGAGA-3'; b45, 5'-GCACTTCC-3'; b46, 5'-GTTACCGA-3'; b47, 5'-GACGTCAC-3'; b48, 5'-GTTACAGC-3'; b49, 5'-TCGATTGC-3'; b50, 5'-GACGATCT-3'; b51, 5'-TGACGCTA-3'; b52, 5'-ATAACGCG-3'; b53, 5'-GATTCACC-3'; b54, 5'-GGCAAGTA-3'; b55, 5'-GATCTAGC-3'; b56, 5'-CATTGCGA-3'; b57, 5'-GCTGTACA-3'; b58, 5'-TAGCAGCT-3'; b59, 5'-AGCGACAT-3'; b60, 5'-CATTAGCC-3'; b61, 5 '-catctgca-3'; b62, 5'-GTCATCGT-3'; b63, 5'-GGACTTCA-3'; b64, 5'-CAGGATCA-3'; b65, 5'-GAACGATG-3'; b66, 5'-CGACTTGC-3'; b67, 5'-GATTCCGT-3' and B68, 5'-CCGCTTAC-3'.
In the invention, the heavy chain variable region gene upstream primer is a single-stranded DNA fragment specifically bound to the heavy chain signal peptide coding gene of the monoclonal antibody, the heavy chain variable region gene downstream index primer is a single-stranded DNA fragment formed by connecting the barcode sequence and a heavy chain variable region gene specific segment, and the heavy chain variable region gene specific segment is DNA specifically bound to the heavy chain signal peptide region and the constant region gene of the monoclonal antibody.
In the invention, the upstream primer of the light chain variable region gene is a single-stranded DNA fragment specifically bound to the light chain signal peptide coding gene of the monoclonal antibody, the downstream index primer of the light chain variable region gene is a single-stranded DNA fragment formed by connecting the barcode sequence and the light chain variable region gene specific section, and the light chain variable region gene specific section is DNA specifically bound to the light chain signal peptide region and the constant region gene of the monoclonal antibody.
In the above method, the hybridoma may be a rat hybridoma or a mouse hybridoma.
The rat hybridoma cells secrete rat antibodies.
The mouse hybridoma cells can secrete mouse antibodies.
In one specific embodiment, the primer composition may be a or B:
A. The hybridoma is a rat hybridoma, the rat antibody heavy chain variable region gene primer composition consists of a rat antibody heavy chain variable region gene upstream primer and a rat heavy chain variable region gene downstream index primer, the rat antibody heavy chain variable region gene upstream primer is 15 single-stranded DNAs of which the nucleotide sequences are respectively 17-31 in a sequence table, and the rat heavy chain variable region gene downstream index primer is a single-stranded DNA of which the nucleotide sequences are 65 in the sequence table; the rat antibody light chain variable region gene primer composition consists of a rat light chain variable region gene upstream primer and a rat light chain variable region gene downstream index primer, wherein the rat light chain variable region gene upstream primer is 15 single-stranded DNAs of which the nucleotide sequences are respectively sequence 1-sequence 15 in a sequence table, and the rat light chain variable region gene downstream index primer is a single-stranded DNA of sequence 64 in the sequence table;
B. the hybridoma is a mouse hybridoma, the mouse antibody heavy chain variable region gene primer composition consists of a mouse antibody heavy chain variable region gene upstream primer and a mouse heavy chain variable region gene downstream index primer, the mouse antibody heavy chain variable region gene upstream primer is 15 single-stranded DNAs with nucleotide sequences of 46-60 in a sequence table respectively, and the mouse heavy chain variable region gene downstream index primer is a single-stranded DNA with nucleotide sequence of 63 in the sequence table; the mouse antibody light chain variable region gene primer composition consists of a mouse light chain variable region gene upstream primer and a mouse light chain variable region gene downstream index primer, wherein the mouse light chain variable region gene upstream primer is 12 single-stranded DNA (deoxyribonucleic acid) of which the nucleotide sequences are respectively 33-44 in a sequence table, and the mouse light chain variable region gene downstream index primer is single-stranded DNA of a sequence 62 in the sequence table.
The invention also provides a composition for amplifying the monoclonal antibody variable region gene, which consists of a heavy chain variable region gene primer composition and a light chain variable region gene primer composition, wherein the heavy chain variable region gene primer composition consists of a heavy chain variable region gene upstream primer and a heavy chain variable region gene downstream index primer, the heavy chain variable region gene downstream index primer consists of N primers, the 5' end of each primer contains a Barcode sequence (sequence specific tag), the other heavy chain variable region gene specific sections are different from each other, and the Barcode sequence of each heavy chain variable region gene downstream index primer is different from each other; the light chain variable region gene primer composition consists of a light chain variable region gene upstream primer and a light chain variable region gene downstream index primer, wherein the light chain variable region gene downstream index primer consists of N primers, the 5' end of each primer contains the Barcode sequence (sequence specific tag), the rest is a light chain variable region gene specific segment, and the Barcode sequence of each light chain variable region gene downstream index primer is different.
In the composition, the barcode sequence is single-stranded DNA for distinguishing different hybridoma cells, and the barcode sequence is any one of 68 single-stranded DNA molecules: b1, 5'-TCGTCATG-3'; b2, 5'-GTCAGTCC-3'; b3, 5'-CGATTACG-3'; b4, 5'-TCTACGAG-3'; b5, 5'-TAACGTGC-3'; b6, 5'-CGAAGGTT-3'; b7, 5'-CACGCATT-3'; b8, 5'-TACCAGGT-3'; b9, 5'-TTACCGAG-3'; b10, 5 '-AGTCACT-3'; b11, 5'-TCGCATTC-3'; b12, 5'-CATGGTAC-3'; b13, 5 '-CACGGGACT-3'; b14, 5'-CTGTAGCA-3'; b15, 5'-AGAGCTTG-3'; b16, 5'-AGCATAGC-3'; b17, 5 '-CCTAACAT-3'; b18, 5'-GTCGTTGA-3'; b19, 5'-TGCGTATC-3'; b20, 5 '-AGCTACC-3'; b21, 5'-GAACGGTA-3'; b22, 5'-GGATTGTC-3'; b23, 5'-TACCGTCT-3'; b24, 5'-CGAGTATC-3'; b25, 5'-TGTACGCT-3'; b26, 5'-TCCTCTGA-3'; b27, 5'-AGCGTATC-3'; b28, 5 '-TCACC-3'; b29, 5'-GACAGTAC-3'; b30, 5 '-GTCCTTC-3'; b31, 5'-GGAGCAAT-3'; b32, 5'-AGGAACAG-3'; b33, 5'-CAGTGCTA-3'; b34, 5 '-CTAGCTAGTAG-3'; b35, 5'-TGCAAGCA-3'; b36, 5'-CGCATCTA-3'; b37, 5'-CGTATCTG-3'; b38, 5'-GTAGACCT-3'; b39, 5'-CGCCATTA-3'; b40, 5'-ATGACACC-3'; b41, 5'-CAAGTTGA-3'; b42, 5'-TGCTGAAG-3'; b43, 5'-ATGACCGT-3'; b44, 5'-TGGCGAGA-3'; b45, 5'-GCACTTCC-3'; b46, 5'-GTTACCGA-3'; b47, 5'-GACGTCAC-3'; b48, 5'-GTTACAGC-3'; b49, 5'-TCGATTGC-3'; b50, 5'-GACGATCT-3'; b51, 5'-TGACGCTA-3'; b52, 5'-ATAACGCG-3'; b53, 5'-GATTCACC-3'; b54, 5'-GGCAAGTA-3'; b55, 5'-GATCTAGC-3'; b56, 5'-CATTGCGA-3'; b57, 5'-GCTGTACA-3'; b58, 5'-TAGCAGCT-3'; b59, 5'-AGCGACAT-3'; b60, 5'-CATTAGCC-3'; b61, 5 '-catctgca-3'; b62, 5'-GTCATCGT-3'; b63, 5'-GGACTTCA-3'; b64, 5'-CAGGATCA-3'; b65, 5'-GAACGATG-3'; b66, 5'-CGACTTGC-3'; b67, 5'-GATTCCGT-3' and B68, 5'-CCGCTTAC-3'.
In the above composition for amplifying a monoclonal antibody variable region gene, the composition may be a or B:
A. the monoclonal antibody is produced by a rat hybridoma, the heavy chain variable region gene primer composition consists of a rat antibody heavy chain variable region gene upstream primer and a rat heavy chain variable region gene downstream index primer, the rat antibody heavy chain variable region gene upstream primer is 15 single-stranded DNAs of which the nucleotide sequences are respectively 17-31 in a sequence table, and the rat heavy chain variable region gene downstream index primer is a single-stranded DNA of which the nucleotide sequences are 65 in the sequence table; the light chain variable region gene primer composition consists of a rat light chain variable region gene upstream primer and a rat light chain variable region gene downstream index primer, wherein the rat light chain variable region gene upstream primer is 15 single-stranded DNAs with nucleotide sequences of sequence 1-sequence 15 in a sequence table respectively, and the rat light chain variable region gene downstream index primer is a single-stranded DNA with sequence 64 in the sequence table;
B. the monoclonal antibody is produced by a mouse hybridoma, the heavy chain variable region gene primer composition consists of an upstream primer of a mouse antibody heavy chain variable region gene and a downstream index primer of the mouse heavy chain variable region gene, and the upstream primer of the mouse antibody heavy chain variable region gene is 15 single-stranded DNA (deoxyribonucleic acid) with nucleotide sequences of 46-60 in a sequence table respectively; the downstream index primer of the mouse heavy chain variable region gene is single-stranded DNA with a nucleotide sequence of sequence 63 in a sequence table; the light chain variable region gene primer composition consists of a mouse light chain variable region gene upstream primer and a mouse light chain variable region gene downstream index primer, wherein the mouse light chain variable region gene upstream primer is 12 single-stranded DNA (deoxyribonucleic acid) with nucleotide sequences of 33-44 in a sequence table respectively; the mouse light chain variable region gene downstream index primer is single-stranded DNA of a sequence 62 in a sequence table.
The invention also provides a kit for amplifying an antibody variable region, which contains the primer composition.
The invention also provides application of the method in high-throughput sequencing of the variable region genes of the monoclonal antibodies.
The invention also provides application of the composition in high-throughput sequencing of monoclonal antibody variable region genes.
The invention also provides application of the kit in preparation of monoclonal antibody variable region gene high-throughput sequencing products.
The invention also provides a device for constructing an antibody evolutionary tree, which comprises:
a PCR amplification device for amplifying variable region genes of monoclonal antibodies secreted by N hybridoma cells by using the composition to obtain antibody variable region gene amplification products; the N is a natural number greater than or equal to 2;
the sequencing device is used for sequencing the gene product comprising the antibody variable region to obtain a sequencing result;
and the analysis device is used for constructing a evolutionary tree of the monoclonal antibody based on the sequencing result.
In the present invention, the sequencing method may be a second generation sequencing (e.g., illumina-Hiseq method).
In the invention, a special label is added to the amplified products of the light chain and heavy chain variable regions of each antibody by adding different barcode sequences (different linkers) on the downstream primers of the variable regions of the antibodies, then the amplified products of the light chain and heavy chain variable regions of N antibodies are mixed, and the mixed amplified products are subjected to second generation sequencing (Illumina-Hiseq). The sequencing result of the mixed product sequence is analyzed by using antibody sequence analysis software which is independently developed by a company, not only can hundreds of antibody variable region sequences be obtained by one-time sequencing, but also the problems of peak sleeving, low-concentration sequencing failure and the like which are frequently encountered in the variable region product generation sequencing (sanger method) are solved, and TA cloning is not needed. Thus, one second generation sequencing (NGS) yields hundreds of antibody light and heavy chain variable region sequences. The sequencing result obtained by the sequencing method is simple to analyze, easy to operate and high in accuracy, TA cloning is not needed to be carried out on the problematic result, and compared with the traditional sequencing method, the sequencing method not only saves manpower and time, but also reduces the sequencing cost.
Drawings
FIG. 1 shows the results of electrophoresis after RNA extraction from different antibodies, lanes A1-A5 are rat antibodies, and lanes A6-A34 are mouse antibodies.
FIG. 2 is a flow chart of an antibody sequence analysis procedure.
FIG. 3 shows the results of light chain variable region gene amplification for different antibodies using primer compositions containing different barcode. Wherein lanes A1-A5 are rat antibodies and lanes A6-A34 are mouse antibodies.
FIG. 4 shows the results of gene amplification of heavy chain variable regions of different antibodies using compositions containing different barcode primers. Wherein lanes A1-A5 are rat antibodies and lanes A6-A34 are mouse antibodies.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The quantitative experiments in the following examples were performed in triplicate unless otherwise indicated.
The DNA polymerase 2×TransTaq (HiFi) PCR SuperMix II (-dye) (Beijing full gold Biotechnology Co., ltd., AS 131-21) of the present invention. Electrophoresis detection and product recovery reagent: agarose (Beijing full gold Biotechnology Co., ltd., GS 201-01); nucleic acid dye (gold of the full formula, GS 101-01); DNA loading buffer (gold of the formula GH 101-01); DNA Marker (full gold, trans2K cube DNA Marker); easy pure cube Quick Gel Extraction Kit (full gold, EG 101-01); animal tissue/cell total RNA extraction kit (day root, DP 451); cell culture medium: DMEM cell culture medium (thermosusher, 11971025); serum: australian foetal calf serum (superfine) (Minhai, SA 102.01); double antibody: penicillin-Streptomycin (Thermo, 15070063); cell culture flask T25 (Thermo, 15070063).
In the nucleotide sequences herein, r=a or G, y=t or C, k=t or G, w=a or T, m=c or a, s=c or G, v=a or C or G, and N is A, T, C or G.
Example 1, establishment of high throughput sequencing of the variable region Gene of rat and mouse antibodies
1. Extraction of cellular RNA
34 hybridoma cells named hybridoma cells A1-A34 are cultured, wherein each hybridoma cell in the 34 hybridoma cells secretes a monoclonal antibody, and the names of the monoclonal antibodies are respectively antibody A1-A34. Wherein the hybridoma cells A1-A5 are rat hybridoma cells, and the hybridoma cells A6-A34 are mouse hybridoma cells. The animal cell culture method comprises the following steps: Hybridoma cells were cultured in medium (1×dmem+10% fetal bovine serum+1% diabody) using T25 cell culture flasks, and the cell culture flasks were placed in a carbon dioxide incubator under culture conditions: 37 ℃,5% CO 2 . To about 5X 10 6 -1×10 7 Cells were recovered in individual/bottle.
RNA is extracted from rat and mouse hybridoma cells by adopting an RNA extraction kit, the extracted RNA is subjected to electrophoresis verification, and the integrity of the RNA is observed by running gel. As a result, as shown in FIG. 1, the total RNA extracted had clear 28S and 18S rRNA bands, and the 28S rRNA band was about 2 times as strong as the 18S rRNA band, suggesting that RNA integrity was better.
cDNA of the hybridoma cells was obtained by reverse transcription using SMART RACE 5'/3' kit (Clontech, cat. No. 634499), and cDNA of hybridoma cells A1-A34 was obtained, respectively.
2. Light chain variable region gene primer and heavy chain variable region gene primer synthesis of rat and mouse antibodies
1) 15 upstream primers of the rat antibody light chain variable region genes are provided, and the specific steps are as follows:
(1) RATKF1:5'-RACATTGTSHTGACYCAGTCTC-3' (sequence 1);
(2) RATKF2:5'-GAAACTGTGATGACCCAGTC-3' (SEQ ID NO: 2);
(3) RATKF3:5'-CAGGCTGTTGTGACTCAGG-3' (SEQ ID NO: 3);
(4) RATKF4:5'-RACRTCCAGWTRACCCAGWCT-3' (sequence 4);
(5) RATKF5:5'-GACATCCAYATGACWCAGWM-3' (SEQ ID NO: 5);
(6) RATKF6:5'-GACATYSRGATGACYMAGTCTC-3' (sequence 6);
(7) RATKF7:5'-GACATTKYGATRACCCARTMT-3' (SEQ ID NO: 7);
(8) RATKF8:5'-GATATTGTGATGACHCARRS-3' (sequence 8);
(9) RATKF9:5'-GATGTTGTTTTGGTGACACA-3' (SEQ ID NO: 9);
(10) RATKF10:5'-GATGTTRTGMTGRCCCAGAC-3' (SEQ ID NO: 10);
(11) RATKF11:5'-GAMATTRTRCTVACCCAGTCT-3' (SEQ ID NO: 11);
(12) RATKF12:5'-GAWAWTGTKCTMMCTCAGTC-3' (SEQ ID NO: 12);
(13) RATKF13:5'-GACGTTGTGCTGACTCAGTC-3' (SEQ ID NO: 13);
(14) RATKF14:5'-CAGCCCGTGCTGCATCAG-3' (SEQ ID NO: 14);
(15) RATKF15:5'-CAGWTCACGCTSACYCARCM-3' (SEQ ID NO: 15).
The downstream primer of the rat antibody light chain variable region gene is index primer, and the general segment (the rat antibody light chain variable region gene specific segment) is RATKR, specifically as follows: RATKR:5 '-ACGTTTYATTTCCARCTKSGTC-3' (SEQ ID NO: 16).
15 upstream primers of heavy chain variable region genes of the rat antibodies are provided, and the specific steps are as follows:
(1) RATHF1:5'-GATCAGGTRCARCTRAMRGAGTCAGG-3' (sequence 17);
(2) RATHF2:5'-GATCAGGTGSAKMTGAAGGAGWC-3' (sequence 18);
(3) RATHF3:5'-GATCARGTGCAGYKGAWGGAGTC-3' (sequence 19);
(4) RATHF4:5'-GATCAGGTHCAGCTGCASCARTCT-3' (sequence 20);
(5) RATHF5:5'-GATCAGATCCAGTTGGYACAGTC-3' (sequence 21);
(6) RATHF6:5'-GATCAGGCCCAGCTGCAGTCTGG-3' (SEQ ID NO: 22);
(7) RATHF7:5'-GATCAGGTCCAGYTGCAGCARTS-3' (sequence 23);
(8) RATHF8:5'-GATCAGATTCAGCTGCARCAGTG-3' (sequence 24);
(9) RATHF9:5'-GATCAAACAGTCCAGCTACAGCAGTC-3' (SEQ ID NO: 25);
(10) RATHF10:5'-GATCAAGARGTCCWGCTGCAKCAGTM-3' (sequence 26);
(11) RATHF11:5'-GATCAGGTTMMTCTGMAASAGTC-3' (SEQ ID NO: 27);
(12) RATHF12:5'-GATCARGTYAASCTRCWGCAGTC-3' (sequence 28);
(13) RATHF13:5'-GATCAAGAGGTAAAGCTGCARCAGTC-3' (sequence 29);
(14) RATHF14:5'-GATCAAGARGTTCARCTGCAGCAGTC-3' (sequence 30);
(15) RATHF15:5'-GATCAAGAGGTGCARMTTCWGGAGWC-3' (SEQ ID NO: 31).
The rat antibody heavy chain variable region gene downstream primer is index primer, and the general segment (rat antibody heavy chain variable region gene specific segment) is RATHR, concretely as follows: RATHR:5'-GATCKGAGGASACGGTGACCRKGG-3' (SEQ ID NO: 32).
The total of 12 upstream primers of the mouse antibody light chain variable region genes are as follows:
(1) Kf1:5'-GCTTACAGGTGCCAGATGT-3' (SEQ ID NO: 33);
(2) MKF2:5'-TCAATTGTAGRTGCCAGATGT-3' (sequence 34);
(3) MKF3:5'-TTACAGTAGGTGTCAGATGT-3' (SEQ ID NO: 35);
(4) Kf4:5'-CAGTCGTAGTTGTCAGATGT-3' (sequence 36);
(5) Kf5:5'-CCTCCTTCTTGGCCAAGA-3' (SEQ ID NO: 37);
(6) MKF6:5'-TTATATGGAGCTGATGGG-3' (SEQ ID NO: 38);
(7) Kf7:5'-GTGTCTGGTGCTCATGGG-3' (SEQ ID NO: 39);
(8) MKF8:5'-CTSTGGTTGTCTGGTGTTGA-3' (sequence 40);
(9) Kf9:5'-GTCTCTGATTCTAGGGCA-3' (SEQ ID NO: 41);
(10) MKF10:5'-TGCTKCKCTGGGTTCCAG-3' (sequence 42);
(11) MKF11:5'-TTGCAGGTGTTGACGGA-3' (SEQ ID NO: 43);
(12) MKF12:5'-GTAGGTGCCTCGTGCAC-3' (SEQ ID NO: 44).
The mouse antibody light chain variable region gene downstream primer is index primer, its general segment (mouse antibody light chain variable region gene specific segment) is MKR, concretely as follows: MKR:5'-CGACTAGTCGACTGGTGGGAAGATGGATACAG-3' (SEQ ID NO: 45).
15 primers upstream of the heavy chain variable region gene of the mouse antibody are provided, and the specific steps are as follows:
(1) MHF1, 5'-ACTGCAGGTRTCCACTCC-3' (SEQ ID NO: 46);
(2) MHF2, 5'-RCTACAGGTGTCCACTCC-3' (SEQ ID NO: 47);
(3) MHF3:5'-ACTGCAGGTGTCCWMTCC-3' (SEQ ID NO: 48);
(4) MHF4, 5'-RCTRCAGGTGTKCACTCC-3' (SEQ ID NO: 49);
(5) MHF5:5'-GCTACAGGTGCTCACTCC-3' (SEQ ID NO: 50);
(6) MHF6:5'-AYTGCAGGTGTCCAYTGC-3' (SEQ ID NO: 51);
(7) MHF7, 5'-GCTAMMGGTGTCCACTTC-3' (SEQ ID NO: 52);
(8) MHF8:5'-RCTRCAGGYGTCCACTCT-3' (SEQ ID NO: 53);
(9) MHF9:5'-CCAAGCTGTATCCTTTCC-3' (SEQ ID NO: 54);
(10) MHF10, 5'-CCAAGCTGTGTCCTRTCC-3' (SEQ ID NO: 55);
(11) MHF11:5'-GTTTTAAAAGGTGTCCTGTG-3' (SEQ ID NO: 56);
(12) MHF12, 5'-CTYTTAAAAGGKGTCCAGWG-3' (sequence 57);
(13) Mhf13:5'-CYTTTAMATGGTATCCAGTGT-3' (sequence 58);
(14) MHF14:5'-CTTTTACATGGTTTCAAGTGT-3' (sequence 59);
(15) MHF15, 5'-YTGTCCCTGCATATGTCYT-3' (SEQ ID NO: 60).
The mouse antibody heavy chain variable region gene downstream primer is index primer, its general segment (mouse antibody heavy chain variable region gene specific segment) is MHR, concretely as follows: MHR 5'-GGATAGACWGATGGGGSTGTYGTT-3' (SEQ ID NO: 61).
3. Barcode sequence and index primer synthesis
The 5 'end of the index primer downstream of the heavy chain variable region gene and the 5' end of the index primer downstream of the light chain variable region gene both contain a Barcode sequence, and the rest is a universal section. The Barcode sequence is a single-stranded DNA used to distinguish between different antibody heavy or light chains. In this example, the Barcode sequence is a single-stranded DNA consisting of 8 nucleotides.
1) Barcode sequence synthesis
The invention provides 68 Barcode sequences of B1-B68, which are specifically shown as follows: b1, 5'-TCGTCATG-3'; b2, 5'-GTCAGTCC-3'; b3, 5'-CGATTACG-3'; b4, 5'-TCTACGAG-3'; b5, 5'-TAACGTGC-3'; b6, 5'-CGAAGGTT-3'; b7, 5'-CACGCATT-3'; b8, 5'-TACCAGGT-3'; b9, 5'-TTACCGAG-3'; b10, 5 '-AGTCACT-3'; b11, 5'-TCGCATTC-3'; b12, 5'-CATGGTAC-3'; b13, 5 '-CACGGGACT-3'; b14, 5'-CTGTAGCA-3'; b15, 5'-AGAGCTTG-3'; b16, 5'-AGCATAGC-3'; b17, 5 '-CCTAACAT-3'; b18, 5'-GTCGTTGA-3'; b19, 5'-TGCGTATC-3'; b20, 5 '-AGCTACC-3'; b21, 5'-GAACGGTA-3'; b22, 5'-GGATTGTC-3'; b23, 5'-TACCGTCT-3'; b24, 5'-CGAGTATC-3'; b25, 5'-TGTACGCT-3'; b26, 5'-TCCTCTGA-3'; b27, 5'-AGCGTATC-3'; b28, 5 '-TCACC-3'; b29, 5'-GACAGTAC-3'; b30, 5 '-GTCCTTC-3'; b31, 5'-GGAGCAAT-3'; b32, 5'-AGGAACAG-3'; b33, 5'-CAGTGCTA-3'; b34, 5 '-CTAGCTAGTAG-3'; b35, 5'-TGCAAGCA-3'; b36, 5'-CGCATCTA-3'; b37, 5'-CGTATCTG-3'; b38, 5'-GTAGACCT-3'; b39, 5'-CGCCATTA-3'; b40, 5'-ATGACACC-3'; b41, 5'-CAAGTTGA-3'; b42, 5'-TGCTGAAG-3'; b43, 5'-ATGACCGT-3'; b44, 5'-TGGCGAGA-3'; b45, 5'-GCACTTCC-3'; b46, 5'-GTTACCGA-3'; b47, 5'-GACGTCAC-3'; b48, 5'-GTTACAGC-3'; b49, 5'-TCGATTGC-3'; b50, 5'-GACGATCT-3'; b51, 5'-TGACGCTA-3'; b52, 5'-ATAACGCG-3'; b53, 5'-GATTCACC-3'; b54, 5'-GGCAAGTA-3'; b55, 5'-GATCTAGC-3'; b56, 5'-CATTGCGA-3'; b57, 5'-GCTGTACA-3'; b58, 5'-TAGCAGCT-3'; b59, 5'-AGCGACAT-3'; b60, 5'-CATTAGCC-3'; b61, 5 '-catctgca-3'; b62, 5'-GTCATCGT-3'; b63, 5'-GGACTTCA-3'; b64, 5'-CAGGATCA-3'; b65, 5'-GAACGATG-3'; b66, 5'-CGACTTGC-3'; b67, 5'-GATTCCGT-3' and B68, 5'-CCGCTTAC-3' are synthesized by general biology.
2) index primer synthesis
The index primer was synthesized by adding the barcode sequence of step 1) above to the 5' end of the antibody heavy chain variable region gene downstream primer or the light chain variable region gene downstream primer. Different barcode sequences can be used as tags to label an antibody light or heavy chain, i.e., one barcode sequence is in one-to-one correspondence with an antibody light or heavy chain variable region gene sequence. Step 1) provides 68 Barcode, and index primer design is specifically as follows:
A. mouse antibody light chain variable region gene downstream index primer:
the 5' -end of the downstream primer universal segment MKR of the mouse antibody light chain is added with a barcode sequence to synthesize a downstream index primer of the mouse antibody light chain variable region gene: 5'-NNNNNNNNCGACTAGTCGACTGGTGGGAAGATGGATACAG-3' (sequence 62 in the sequence listing). The nucleotide sequence of the 1 st to 8 th (5 '-NNNNNN-3') of the sequence 62 is Barcode, and the nucleotide sequence of 68 Barcode is specifically shown in the step 1). In this step, 29 different barcode sequences were used in total for B1-B29 in the above step 1), and 29 mouse antibody light chain variable region gene downstream index primers were obtained.
B. Mouse antibody heavy chain variable region gene downstream index primer:
adding a barcode sequence into the 5' end of a universal section MHR of a downstream primer of a heavy chain of the mouse antibody to synthesize an index primer downstream of a variable region gene of the heavy chain of the mouse antibody: 5'-NNNNNNNNGGATAGACWGATGGGGSTGTYGTT-3' (sequence 63 in the sequence listing). The 1 st to 8 th (5 '-NNNNNNNN-3') of the sequence 63 is Barcode, and 29 different Barcode sequences are used in the step 1) in total in the step B30-B58, so that 29 mouse antibody heavy chain variable region gene downstream index primers containing the different Barcode sequences are obtained.
C. Rat antibody light chain variable region gene downstream index primer:
adding a barcode sequence into the 5' -end of a general segment RATKR of a downstream primer of a light chain of the rat antibody to synthesize a downstream index primer of a variable region gene of the light chain of the rat antibody: 5'-NNNNNNNNACGTTTYATTTCCARCTKSGTC-3' (sequence 64 in the sequence listing). The nucleotide sequence of the 1 st to 8 th (5 '-NNNNNN-3') of the sequence 64 is Barcode, and the nucleotide sequence of 68 Barcode is specifically shown in the step 1). In this step, 5 different barcode sequences were used in total for B59-B63 in the above step 1), and 5 rat antibody light chain variable region gene downstream index primers containing the different barcode sequences were obtained.
D. Rat antibody heavy chain variable region gene downstream index primer:
adding a barcode sequence into the 5' -end of a general segment RATHR of a downstream primer of a heavy chain of the rat antibody to synthesize an index primer downstream of a variable region gene of the heavy chain of the rat antibody: 5'-NNNNNNNNGATCKGAGGASACGGTGACCRKGG-3' (sequence 65 in the sequence listing). The nucleotide sequence of the 68 Barcode at positions 1-8 (5 '-NNNNNNNN-3') of the sequence 65 is specifically shown in the step 1). In this step, 5 different barcode sequences were used in total for B64-B68 in the above step 1), and 5 rat antibody heavy chain variable region gene downstream index primers containing the different barcode sequences were obtained.
4. High-throughput sequencing method establishment of variable region of rat and mouse antibodies
1) Mixing of amplification primers
The primer powder obtained in steps 2 and 3 was centrifuged at 12000rpm for 10min using a centrifuge before dissolving the primer. The solution was dissolved using enzyme-free water.
15 kinds of the upstream primers of the rat antibody light chain variable region gene in the step 2 are mixed to obtain LRmix, wherein the content of the upstream primer of each rat antibody light chain variable region gene in the LRmix is 10 mu mol/L.
15 kinds of the upstream primers of the heavy chain variable region genes of the rat antibodies in the step 2 are mixed to obtain HR mix, wherein the content of the upstream primer of each heavy chain variable region gene of the rat antibodies in the HR mix is 10 mu mol/L.
Mixing 12 kinds of the upstream primers of the light chain variable region genes of the mouse antibodies obtained in the step 2 to obtain LMmix, wherein the content of the upstream primer of each light chain variable region gene of the mouse antibodies in the LMmix is 10 mu mol/L.
15 kinds of the primers upstream of the heavy chain variable region gene of the mouse antibody in the step 2 are mixed to obtain HMmix, and the content of the upstream primer of the heavy chain variable region gene of each mouse antibody in the HMmix is 10 mu mol/L.
2) PCR amplification reaction
A. Antibody heavy chain variable region gene PCR system:
the difference between the rat antibody heavy chain variable region gene PCR system and the mouse antibody heavy chain variable region PCR system is only that: the cDNA template, upstream primer and downstream index primer were different, and the remainder were identical.
The rat antibody heavy chain variable region gene PCR system is divided into 5 PCR systems, and the 5 PCR systems are named as an A1 rat antibody heavy chain variable region gene PCR system, an A2 rat antibody heavy chain variable region gene PCR system, an A3 rat antibody heavy chain variable region gene PCR system, an A4 rat antibody heavy chain variable region gene PCR system and an A5 rat antibody heavy chain variable region gene PCR system respectively. The upstream primers of the 5 PCR systems are HR mix; the cDNA and downstream index primers of these 5 PCR systems were different, wherein: the cDNA in the A1 rat antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A1, the cDNA in the downstream index primer contains a Barcode sequence of B1 and 5'-TCGTCATG-3', the cDNA in the A2 rat antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A2, the cDNA in the downstream index primer contains a Barcode sequence of B2 and 5'-GTCAGTCC-3', the cDNA in the A3 rat antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A3, the cDNA in the downstream index primer contains a Barcode sequence of B3 and 5'-CGATTACG-3', the cDNA in the A4 rat antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A4, the cDNA in the downstream index primer contains a Barcode sequence of B4 and 5'-TCTACGAG-3', the cDNA in the A5 rat antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A5, and the cDNA in the downstream index primer contains a Barcode sequence of B3 and 5 '-TATACG-3'.
Rat antibody heavy chain variable region gene PCR system: 1 mu L of cDNA of 1 hybridoma cell among the A1-A5 hybridoma cells in step 1, 1 mu L of heavy chain upstream primer (HR mix) of the rat antibody, 1 mu L of downstream index primer (containing specific Barcode sequence in one-to-one correspondence with cDNA of different hybridoma cells), 2X Trans Taq HiFi PCR Super Mix II 5 mu L, ddH 2 O2. Mu.L, total volume was 10. Mu.L.
The heavy chain variable region gene PCR system of the mouse antibody has 29 PCR systems, these 5 PCR systems are designated as the A6 mouse antibody heavy chain variable region gene PCR system, the A7 mouse antibody heavy chain variable region gene PCR system, the A8 mouse antibody heavy chain variable region gene PCR system, the A9 mouse antibody heavy chain variable region gene PCR system, the A10 mouse antibody heavy chain variable region gene PCR system, the A11 mouse antibody heavy chain variable region gene PCR system, the A12 mouse antibody heavy chain variable region gene PCR system, the A13 mouse antibody heavy chain variable region gene PCR system, the A14 mouse antibody heavy chain variable region PCR system, the A15 mouse antibody heavy chain variable region gene PCR system, the A16 mouse antibody heavy chain variable region PCR system, the A17 mouse antibody heavy chain variable region PCR system, the A18 mouse heavy chain variable region PCR system, the A19 mouse heavy chain variable region PCR system, the A18 mouse heavy chain variable region PCR system A20 mouse antibody heavy chain variable region gene PCR system, A21 mouse antibody heavy chain variable region gene PCR system, A22 mouse antibody heavy chain variable region gene PCR system, A23 mouse antibody heavy chain variable region gene PCR system, A24 mouse antibody heavy chain variable region gene PCR system, A25 mouse antibody heavy chain variable region gene PCR system, A26 mouse antibody heavy chain variable region gene PCR system, A27 mouse antibody heavy chain variable region gene PCR system, A28 mouse antibody heavy chain variable region gene PCR system, A29 mouse antibody heavy chain variable region gene PCR system, A30 mouse antibody heavy chain variable region gene PCR system, A31 mouse antibody heavy chain variable region gene PCR system, A32 mouse antibody heavy chain variable region gene PCR system, A33 mouse antibody heavy chain variable region PCR system, A34 mouse antibody heavy chain variable region gene PCR system.
The upstream primers of the 29 PCR systems are HMmix; the cDNA and downstream index primers of these 29 PCR systems were all different, wherein: the cDNA in the A6 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A6, the cDNA in the downstream index primer contains Barcode sequence B6 and 5' -CGAAGGTT-3', the cDNA in the A7 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A7, the cDNA in the downstream index primer contains Barcode sequence B7 and 5' -CACGCATT-3', the cDNA in the A8 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A8, the cDNA in the downstream index primer contains Barcode sequence B8 and 5' -TACCAGGT-3', the cDNA in the A9 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A9, the cDNA in the downstream index primer contains Barcode sequence B9 and 5' -TTACCGAG-3', the cDNA in the A10 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A10, and the downstream index primer contains Barcode sequence B10 and the cDNA of the CCTAAGGT-3 ', the cDNA of the downstream index primer contains the cDNA of the hybridoma cell A9; the cDNA in the A11 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A11, the cDNA in the downstream index primer contains a Barcode sequence of B11, 5' -TCGCATTC-3', A12 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A12, the cDNA in the downstream index primer contains a Barcode sequence of B12, 5' -CATGGTAC-3', A13 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A13, the cDNA in the downstream index primer contains a Barcode sequence of B13, 5' -CACGGAT-3 ', A14 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A14, the cDNA in the downstream index primer contains a Barcode sequence of B14, 5' -CTGTAGCA-3', A15 mouse antibody heavy chain variable region system is the cDNA of the hybridoma cell A15, and the cDNA in the downstream index primer contains a Barcode sequence of B13, 5' -CTAGACT-3 ', A14 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A15, the cDNA in the A16 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A16, the cDNA in the downstream index primer contains Barcode sequence B16 and 5'-AGCATAGC-3', the cDNA in the A17 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A17, the cDNA in the downstream index primer contains Barcode sequence B17 and 5 '-CCTACAT-3', the cDNA in the A18 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A18, the cDNA in the downstream index primer contains Barcode sequence B18 and 5 '-GTCGTTTT-3', the cDNA in the A19 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A19, the cDNA in the downstream index primer contains Barcode sequence B19 and 5 '-CGTATC-3', the cDNA in the A20 mouse antibody heavy chain variable region PCR system is the hybridoma cell A20, and the cDNA in the downstream index primer contains Barcode sequence B20 and the cDNA in the CTA 3 '-CTA 3' to be the cDNA; the cDNA in the A21 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A21, the cDNA in the downstream index primer contains Barcode sequence B21 and 5'-GAACGGTA-3', the cDNA in the A22 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A22, the cDNA in the downstream index primer contains Barcode sequence B22 and 5'-GGATTGTC-3', the cDNA in the A23 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A23, the cDNA in the downstream index primer contains Barcode sequence B23 and 5'-TACCGTCT-3', the cDNA in the A24 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A24, the cDNA in the downstream index primer contains Barcode sequence B24 and 5'-CGAGTATC-3', the cDNA in the A25 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A25, and the downstream index primer contains Barcode sequence B23 and 5'-TACCGTCT-3', the cDNA in the downstream index primer contains the cDNA of the B24, the cDNA in the A16 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A26, the cDNA in the downstream index primer contains a Barcode sequence of B26 and 5'-TCCTCTGA-3', the cDNA in the A27 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A27, the cDNA in the downstream index primer contains a Barcode sequence of B27 and 5'-AGCGTATC-3', the cDNA in the A28 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A28, the cDNA in the downstream index primer contains a Barcode sequence of B28 and 5 '-TCACC-3', the cDNA in the A29 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A29, the cDNA in the downstream index primer contains a Barcode sequence of B29 and 5'-GACAGTAC-3', the cDNA in the A30 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A30, the downstream index primer contains a Barcode sequence of B30 and 5 '-GTCCTAC-3', the cDNA in the A31 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A31, the downstream index primer contains a Barcode sequence of B31 and 5'-GGAGCAAT-3', the cDNA in the A32 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A32, the downstream index primer contains a Barcode sequence of B32 and 5'-AGGAACAG-3', the cDNA in the A33 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A33, the downstream index primer contains a Barcode sequence of B33 and 5'-CAGTGCTA-3', the cDNA in the A34 mouse antibody heavy chain variable region PCR system is the cDNA of the hybridoma cell A34, and the downstream index primer contains a Barcode sequence of B34 and 5 '-CTAGGC3'.
Mouse antibody heavy chain variable region gene PCR system: 1 mu L of cDNA of 1 hybridoma cell among the A6-A34 hybridoma cells in step 1, 1 mu L of mouse antibody heavy chain upstream primer (HMmix), 1 mu L of downstream index primer (containing specific Barcode sequence in one-to-one correspondence with cDNA of different hybridoma cells), 2X Trans Taq HiFi PCR Super Mix II 5 mu L, ddH 2 O2. Mu.L, total volume was 10. Mu.L.
B. Antibody light chain variable region gene PCR system:
the difference between the rat antibody light chain variable region PCR system and the mouse antibody light chain variable region PCR system is only that: the cDNA template, upstream primer and downstream index primer were different, and the remainder were identical.
The rat antibody light chain variable region gene PCR system is divided into 5 PCR systems, and the 5 PCR systems are named as an A1 rat antibody light chain variable region gene PCR system, an A2 rat antibody light chain variable region gene PCR system, an A3 rat antibody light chain variable region gene PCR system, an A4 rat antibody light chain variable region gene PCR system and an A5 rat antibody light chain variable region gene PCR system respectively. The upstream primers of the 5 PCR systems are all LRmix; the cDNA and downstream index primers of these 5 PCR systems were different, wherein: the cDNA in the A1 rat antibody light chain variable region PCR system is the cDNA of the hybridoma cell A1, the cDNA in the downstream index primer contains a Barcode sequence of B35, 5' -TGCAAGCA-3', the cDNA in the A2 rat antibody light chain variable region PCR system is the cDNA of the hybridoma cell A2, the cDNA in the downstream index primer contains a Barcode sequence of B36, 5' -CGCATCTA-3', the cDNA in the A3 rat antibody light chain variable region PCR system is the cDNA of the hybridoma cell A3, the cDNA in the downstream index primer contains a Barcode sequence of B37, 5' -CGTATCTG-3', the cDNA in the A4 rat antibody light chain variable region PCR system is the cDNA of the hybridoma cell A4, the cDNA in the downstream index primer contains a Barcode sequence of B38, 5' -GTAGCT-3 ', the cDNA in the A5 rat antibody light chain variable region system is the cDNA of the hybridoma cell A5, and the cDNA in the downstream index primer contains a Barcode sequence of B39-3 '.
Rat antibody light chain variable region gene PCR system: 1 mu L of cDNA of 1 hybridoma cell among the A1-A5 hybridoma cells in step 1, 1 mu L of rat antibody light chain upstream primer (LRmix), 1 mu L of downstream index primer (containing specific Barcode sequence in one-to-one correspondence with cDNA of different hybridoma cells), 2X Trans Taq HiFi PCR Super Mix II 5 mu L, ddH 2 O2. Mu.L, total volume was 10. Mu.L.
The mouse antibody light chain variable region gene PCR system has 29 PCR systems, these 5 PCR systems are designated as the A6 mouse antibody light chain variable region gene PCR system, the A7 mouse antibody light chain variable region gene PCR system, the A8 mouse antibody light chain variable region gene PCR system, the A9 mouse antibody light chain variable region gene PCR system, the A10 mouse antibody light chain variable region gene PCR system, the A11 mouse antibody light chain variable region gene PCR system, the A12 mouse antibody light chain variable region gene PCR system, the A13 mouse antibody light chain variable region gene PCR system, the A14 mouse antibody light chain variable region PCR system, the A15 mouse antibody light chain variable region gene PCR system, the A16 mouse antibody light chain variable region PCR system, the A17 mouse antibody light chain variable region PCR system, the A18 mouse light chain variable region PCR system, the A19 mouse antibody light chain variable region PCR system, the A18 mouse antibody light chain variable region PCR system A20 mouse antibody light chain variable region gene PCR system, A21 mouse antibody light chain variable region gene PCR system, A22 mouse antibody light chain variable region gene PCR system, A23 mouse antibody light chain variable region gene PCR system, A24 mouse antibody light chain variable region gene PCR system, A25 mouse antibody light chain variable region gene PCR system, A26 mouse antibody light chain variable region gene PCR system, A27 mouse antibody light chain variable region gene PCR system, A28 mouse antibody light chain variable region gene PCR system, A29 mouse antibody light chain variable region gene PCR system, A30 mouse antibody light chain variable region gene PCR system, A31 mouse antibody light chain variable region gene PCR system, A32 mouse antibody light chain variable region gene PCR system, A33 mouse antibody light chain variable region PCR system, A34 mouse antibody light chain variable region gene PCR system.
The upstream primers of the 29 PCR systems are LMmix; the cDNA and downstream index primers of these 29 PCR systems were all different, wherein: the cDNA in the A6 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A6, the cDNA in the downstream index primer contains a Barcode sequence of B40, 5'-ATGACACC-3', A7 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A7, the cDNA in the downstream index primer contains a Barcode sequence of B41, 5'-CAAGTTGA-3', A8 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A8, the cDNA in the downstream index primer contains a Barcode sequence of B42, 5'-TGCTGAAG-3', A9 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A9, the cDNA in the downstream index primer contains a Barcode sequence of B43, 5'-ATGACCGT-3', A10 mouse antibody light chain variable region system is the cDNA of the hybridoma cell A10, and the cDNA in the downstream index primer contains a Barcode sequence of B42, 5 '-TGGAAG-3', A9 and GCGAG-3 'are the cDNA; the cDNA in the A11 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A11, the cDNA in the downstream index primer contains Barcode sequence B45 and 5' -GCACTTCC-3', the cDNA in the A12 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A12, the cDNA in the downstream index primer contains Barcode sequence B46 and 5' -GTTACCGA-3', the cDNA in the A13 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A13, the cDNA in the downstream index primer contains Barcode sequence B47 and 5' -GACGTCAC-3', the cDNA in the A14 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A14, the cDNA in the downstream index primer contains Barcode sequence B48 and 5' -GTTACAGC-3', the cDNA in the A15 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A15, and the cDNA in the downstream index primer contains Barcode sequence B47 and 5' -GACGTCTCAC-3 ', the cDNA in the downstream index primer contains the cDNA of the TTGC-3', the cDNA, the cDNA in the A16 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A16, the cDNA in the downstream index primer contains Barcode sequence B50 and 5'-GACGATCT-3', the cDNA in the A17 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A17, the cDNA in the downstream index primer contains Barcode sequence B51 and 5'-TGACGCTA-3', the cDNA in the A18 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A18, the cDNA in the downstream index primer contains Barcode sequence B52 and 5 '-ATGCG-3', the cDNA in the A19 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A19, the Barcode sequence contained in the downstream index primer contains B53 and 5'-GATTCACC-3', the cDNA in the A20 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A20, and the downstream index primer contains Barcode sequence B54 and 5 '-GACAGCG-3', the cDNA contained in the downstream index primer contains the cDNA of the AACCTA3 '; the cDNA in the A21 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A21, the Barcode sequence contained in the downstream index primer is B55, 5' -GATCTAGC-3', the cDNA in the A22 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A22, the Barcode sequence contained in the downstream index primer is B56, 5' -CATTGCGA-3', the cDNA in the A23 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A23, the Barcode sequence contained in the downstream index primer is B57, 5' -GCTGTACA-3', the cDNA in the A24 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A24, the Barcode sequence contained in the downstream index primer is B58, 5' -CAGCT-3 ', the cDNA in the A25 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A25, and the downstream index primer is the cDNA of the Barcode sequence of the A59, the downstream index primer is the cDNA of the ACA 3', the ACA 24, the cDNA in the A26 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A26, the cDNA in the downstream index primer contains a Barcode sequence of B60 and 5'-CATTAGCC-3', the cDNA in the A27 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A27, the cDNA in the downstream index primer contains a Barcode sequence of B61 and 5'-CATCTCCA-3', the cDNA in the A28 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A28, the cDNA in the downstream index primer contains a Barcode sequence of B62 and 5'-GTCATCGT-3', the cDNA in the A29 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A29, the cDNA in the downstream index primer contains a Barcode sequence of B63 and 5'-GGACTTCA-3', the cDNA in the A30 mouse antibody light chain variable region PCR system is the cDNA of the hybridoma cell A30, the downstream index primer contains Barcode sequence B64, 5'-CAGGATCA-3', cDNA in A31 mouse antibody light chain variable region PCR system is cDNA of hybridoma cell A31, the downstream index primer contains Barcode sequence B65, 5'-GAACGATG-3', cDNA in A32 mouse antibody light chain variable region PCR system is cDNA of hybridoma cell A32, the downstream index primer contains Barcode sequence B66, 5'-CGACTTGC-3', cDNA in A33 mouse antibody light chain variable region PCR system is cDNA of hybridoma cell A33, the downstream index primer contains Barcode sequence B67, 5'-GATTCCGT-3', cDNA in A34 mouse antibody light chain variable region PCR system is cDNA of hybridoma cell A34, and the downstream index primer contains Barcode sequence B68, 5 '-CCTTGCAC-3'.
Mouse antibody light chain variable region gene PCR system: 1 mu L of cDNA of 1 hybridoma cell among the A6-A34 hybridoma cells in step 1, 1 mu L of mouse antibody light chain upstream primer (LMmix), 1 mu L of downstream index primer (containing specific Barcode sequence corresponding to cDNA of different hybridoma cells one by one), 2X Trans Taq HiFi PCR Super Mix II 5 mu L, ddH 2 O2. Mu.L, total volume was 10. Mu.L.
C. The reaction conditions of the PCR system are as follows: pre-denaturation at 94 ℃ for 5min; the following 30 cycles were then performed: denaturation at 94℃for 30sec, annealing at 55℃for 30sec, elongation at 72℃for 30sec; then extending at 72 ℃ for 7min and preserving at 4 ℃.
3) Detection of amplification products: mu.L of the PCR product obtained in step 2) of the present sequencing method was taken and detected by 1.5% agarose gel electrophoresis.
4) Sequencing analysis: mixing the heavy chain variable region gene PCR products and the light chain variable region gene PCR products of all antibodies to be tested, and carrying out product recovery by using a gel recovery kit. And (5) delivering the recovered product to a norstanding grain source for high-throughput sequencing.
5) Data analysis: the sequencing result data obtained in the step 4) are analyzed by using an antibody sequence analysis program which is developed by a company. The flow chart of the antibody sequence analysis procedure is shown in FIG. 2. The design idea of the data analysis program is as follows:
A. Basic structure of antibody sequences
(1) The heavy chain of the antibody from the N end to the C end is a variable region formed by connecting one of VH sequences in a plurality of genomes, one of DH sequences in a plurality of genomes and one of JH sequences in a plurality of genomes through a middle randomly generated Junction1H sequence and a Junction2H sequence, so as to form a VH-Junction1H-DH-Junction 2H-Junction with the following structure;
(2) The antibody light chain is formed by connecting variable regions formed by connecting one of VL sequences in a plurality of genomes and one of JLs in a plurality of genomes from the N end to the C end through a connecting randomly generated Junction L sequence, and the VL-Junction L-JL with the following structure is formed.
B. Heavy chain sequence analysis concept
(1) Establishing a heavy chain database VH, DH and JH;
(2) C-terminal sequences are compared with a database JH to determine JH fragments;
(3) Comparing the N-terminal sequence with a database VH to determine a VH fragment;
(4) Removing the JH fragment and the VH fragment, comparing the rest sequences with a database DH, and determining the DH fragment, and randomly generated Junction1H sequences and Junction2H at two ends.
C. Light chain sequence analysis concept
(1) Establishing a light chain database VL, JL;
(2) C-terminal sequences are compared with a database JL to determine JL fragments;
(3) Comparing the N-terminal sequence with a database VL to determine a VL fragment;
(4) The JL and VL fragments were removed to yield randomly generated junction l sequences.
D. Tracking heavy chain light chain evolution
Since the heavy and light chains of antibodies are constantly mutated during immunization, the evolution of antibodies can be followed. The method comprises the following specific steps:
(1) Extracting heavy chain sequences with the same basic structure, namely sequences with the same VH fragment, DH fragment and JH fragment, and establishing a heavy chain evolutionary tree by using a genome sequence without mutation as an origin through a Clustalomega program;
(2) Extracting light chain sequences with the same basic structure, namely sequences with the same VL fragments and JL fragments, and establishing a light chain evolutionary tree by using a Clustalomega program by taking a genome sequence without mutation as an origin.
5 mouse antibodies, 29 mouse antibodies were amplified using the Barcode-containing primers, and the results are shown in figures 3 and 4: the antibody light chain variable region gene and the heavy chain variable region gene are amplified by using the primer containing the barcode, and the PCR products amplify bands at 250bp-500bp, wherein the sizes of the bands are consistent with the predicted sizes.
The PCR products containing barcode were mixed and subjected to second generation sequencing. Sequencing results correct light and heavy chain variable region gene sequences were obtained for 5 rat antibodies and 29 mouse antibodies using software analysis.
The antibody sequencing results were as follows: all antibodies were sequenced successfully. The nucleotide sequence of the detailed sequencing result is shown as a sequence 66-sequence 133 in a sequence table. Wherein: the light chain variable region gene sequence of the antibody A1 is a DNA molecule of a sequence 66 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 67 in the sequence table; the light chain variable region gene sequence of the antibody A2 is a DNA molecule of a sequence 68 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 69 in the sequence table; the light chain variable region gene sequence of the antibody A3 is a DNA molecule of a sequence 70 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 71 in the sequence table; the light chain variable region gene sequence of the antibody A4 is a DNA molecule of a sequence 72 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 73 in the sequence table; the light chain variable region gene sequence of the antibody A5 is a DNA molecule with a sequence 74 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule with a sequence 75 in the sequence table; the light chain variable region gene sequence of the antibody A6 is a DNA molecule of a sequence 76 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 77 in the sequence table; the light chain variable region gene sequence of the antibody A7 is a DNA molecule of a sequence 78 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 79 in the sequence table; the light chain variable region gene sequence of the antibody A8 is a DNA molecule of a sequence 80 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 81 in the sequence table; the light chain variable region gene sequence of the antibody A9 is a DNA molecule of a sequence 82 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 83 in the sequence table; the light chain variable region gene sequence of the antibody A10 is a DNA molecule of a sequence 84 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 85 in the sequence table; the light chain variable region gene sequence of the antibody A11 is a DNA molecule of a sequence 86 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 87 in the sequence table; the light chain variable region gene sequence of the antibody A12 is a DNA molecule of a sequence 88 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 89 in the sequence table; the light chain variable region gene sequence of the antibody A13 is a DNA molecule of a sequence 90 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 91 in the sequence table; the light chain variable region gene sequence of the antibody A14 is a DNA molecule of a sequence 92 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 93 in the sequence table; the light chain variable region gene sequence of the antibody A15 is a DNA molecule of a sequence 94 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 95 in the sequence table; the light chain variable region gene sequence of the antibody A16 is a DNA molecule of a sequence 96 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 97 in the sequence table; the light chain variable region gene sequence of the antibody A17 is a DNA molecule of a sequence 98 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 99 in the sequence table; the light chain variable region gene sequence of the antibody A18 is a DNA molecule of a sequence 100 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 101 in the sequence table; the light chain variable region gene sequence of the antibody A19 is a DNA molecule of a sequence 102 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 103 in the sequence table; the light chain variable region gene sequence of the antibody A20 is a DNA molecule of a sequence 104 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 105 in the sequence table; the light chain variable region gene sequence of the antibody A21 is a DNA molecule of a sequence 106 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 107 in the sequence table; the light chain variable region gene sequence of the antibody A22 is a DNA molecule of a sequence 108 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 109 in the sequence table; the light chain variable region gene sequence of the antibody A23 is a DNA molecule of a sequence 110 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 111 in the sequence table; the light chain variable region gene sequence of the antibody A24 is a DNA molecule of a sequence 112 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 113 in the sequence table; the light chain variable region gene sequence of the antibody A25 is a DNA molecule of a sequence 114 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 115 in the sequence table; the light chain variable region gene sequence of the antibody A26 is a DNA molecule of a sequence 116 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 117 in the sequence table; the light chain variable region gene sequence of the antibody A27 is a DNA molecule of a sequence 118 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 119 in the sequence table; the light chain variable region gene sequence of the antibody 28 is a DNA molecule of a sequence 120 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 121 in the sequence table; the light chain variable region gene sequence of the antibody A29 is a DNA molecule of a sequence 122 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 123 in the sequence table; the light chain variable region gene sequence of the antibody A30 is a DNA molecule of a sequence 124 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 125 in the sequence table; the light chain variable region gene sequence of the antibody A31 is a DNA molecule of a sequence 126 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 127 in the sequence table; the light chain variable region gene sequence of the antibody A32 is a DNA molecule of a sequence 128 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 129 in the sequence table; the light chain variable region gene sequence of the antibody A33 is a DNA molecule of a sequence 130 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 131 in the sequence table; the light chain variable region gene sequence of the antibody A34 is a DNA molecule of a sequence 132 in a sequence table, and the heavy chain variable region gene sequence is a DNA molecule of a sequence 133 in the sequence table.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (10)
1. A method for high throughput sequencing of a monoclonal antibody variable region gene, said method comprising the steps of:
1) Obtaining cDNA of N kinds of hybridoma cells, wherein each hybridoma cell in the N kinds of hybridoma cells secretes a monoclonal antibody, and N is a natural number greater than or equal to 2;
2) Using cDNA of each hybridoma cell as a template, and respectively carrying out PCR amplification on a heavy chain variable region gene and a light chain variable region gene of the monoclonal antibody secreted by each hybridoma cell by using a heavy chain variable region gene primer composition and a light chain variable region gene primer composition to obtain a heavy chain variable region gene PCR product of the monoclonal antibody secreted by each hybridoma cell and a light chain variable region gene PCR product of the monoclonal antibody secreted by each hybridoma cell; mixing and sequencing heavy chain variable region gene PCR products of the monoclonal antibodies secreted by the N hybridoma cells to obtain sequencing results of the heavy chain variable region gene PCR products of the monoclonal antibodies secreted by the N hybridoma cells; mixing and sequencing the light chain variable region gene PCR products of the monoclonal antibodies secreted by the N hybridoma cells to obtain the sequencing result of the light chain variable region gene PCR products of the monoclonal antibodies secreted by the N hybridoma cells;
The heavy chain variable region gene primer composition consists of a heavy chain variable region gene upstream primer and a heavy chain variable region gene downstream index primer, wherein the heavy chain variable region gene downstream index primer consists of N primers, the 5' end of each primer contains a Barcode sequence, the other heavy chain variable region gene specific sections, and the Barcode sequence of each heavy chain variable region gene downstream index primer is different;
the light chain variable region gene primer composition consists of a light chain variable region gene upstream primer and a light chain variable region gene downstream index primer, wherein the light chain variable region gene downstream index primer consists of N primers, the 5' end of each primer contains the Barcode sequence, the rest is a light chain variable region gene specific section, and the Barcode sequence of each light chain variable region gene downstream index primer is different;
3) And 2) carrying out data analysis on the sequencing result of the heavy chain variable region gene PCR products of the monoclonal antibodies secreted by the N hybridoma cells obtained in the 2) and the sequencing result of the light chain variable region gene PCR products of the monoclonal antibodies secreted by the N hybridoma cells, so as to respectively obtain the sequences of the monoclonal antibody variable region genes secreted by each hybridoma cell.
2. The method of claim 1, wherein the barcode sequence is a single-stranded DNA for distinguishing between different hybridoma cells, the barcode sequence being any one of 68 single-stranded DNA molecules: b1, 5'-TCGTCATG-3'; b2, 5'-GTCAGTCC-3'; b3, 5'-CGATTACG-3'; b4, 5'-TCTACGAG-3'; b5, 5'-TAACGTGC-3'; b6, 5'-CGAAGGTT-3'; b7, 5'-CACGCATT-3'; b8, 5'-TACCAGGT-3'; b9, 5'-TTACCGAG-3'; b10, 5 '-AGTCACT-3'; b11, 5'-TCGCATTC-3'; b12, 5'-CATGGTAC-3'; b13, 5 '-CACGGGACT-3'; b14, 5'-CTGTAGCA-3'; b15, 5'-AGAGCTTG-3'; b16, 5'-AGCATAGC-3'; b17, 5 '-CCTAACAT-3'; b18, 5'-GTCGTTGA-3'; b19, 5'-TGCGTATC-3'; b20, 5 '-AGCTACC-3'; b21, 5'-GAACGGTA-3'; b22, 5'-GGATTGTC-3'; b23, 5'-TACCGTCT-3'; b24, 5'-CGAGTATC-3'; b25, 5'-TGTACGCT-3'; b26, 5'-TCCTCTGA-3'; b27, 5'-AGCGTATC-3'; b28, 5 '-TCACC-3'; b29, 5'-GACAGTAC-3'; b30, 5 '-GTCCTTC-3'; b31, 5'-GGAGCAAT-3'; b32, 5'-AGGAACAG-3'; b33, 5'-CAGTGCTA-3'; b34, 5 '-CTAGCTAGTAG-3'; b35, 5'-TGCAAGCA-3'; b36, 5'-CGCATCTA-3'; b37, 5'-CGTATCTG-3'; b38, 5'-GTAGACCT-3'; b39, 5'-CGCCATTA-3'; b40, 5'-ATGACACC-3'; b41, 5'-CAAGTTGA-3'; b42, 5'-TGCTGAAG-3'; b43, 5'-ATGACCGT-3'; b44, 5'-TGGCGAGA-3'; b45, 5'-GCACTTCC-3'; b46, 5'-GTTACCGA-3'; b47, 5'-GACGTCAC-3'; b48, 5'-GTTACAGC-3'; b49, 5'-TCGATTGC-3'; b50, 5'-GACGATCT-3'; b51, 5'-TGACGCTA-3'; b52, 5'-ATAACGCG-3'; b53, 5'-GATTCACC-3'; b54, 5'-GGCAAGTA-3'; b55, 5'-GATCTAGC-3'; b56, 5'-CATTGCGA-3'; b57, 5'-GCTGTACA-3'; b58, 5'-TAGCAGCT-3'; b59, 5'-AGCGACAT-3'; b60, 5'-CATTAGCC-3'; b61, 5 '-catctgca-3'; b62, 5'-GTCATCGT-3'; b63, 5'-GGACTTCA-3'; b64, 5'-CAGGATCA-3'; b65, 5'-GAACGATG-3'; b66, 5'-CGACTTGC-3'; b67, 5'-GATTCCGT-3' and B68, 5'-CCGCTTAC-3'.
3. The method of claim 1, wherein the heavy chain variable region gene upstream primer is a single-stranded DNA fragment specifically binding to a heavy chain signal peptide-encoding gene of the monoclonal antibody, the heavy chain variable region gene downstream index primer is a single-stranded DNA fragment formed by ligating the barcode sequence and a heavy chain variable region gene-specific segment, which is DNA specifically binding to a heavy chain signal peptide region and a constant region gene of the monoclonal antibody; the upstream primer of the light chain variable region gene is a single-stranded DNA fragment specifically combined with the light chain signal peptide coding gene of the monoclonal antibody, the downstream index primer of the light chain variable region gene is a single-stranded DNA fragment formed by connecting the barcode sequence and a light chain variable region gene specific section, and the light chain variable region gene specific section is DNA specifically combined with the light chain signal peptide region of the monoclonal antibody and a constant region gene.
4. A method according to claim 1, 2 or 3, wherein the primer composition is a or B:
A. the hybridoma is a rat hybridoma, the heavy chain variable region gene primer composition consists of a rat antibody heavy chain variable region gene upstream primer and a rat heavy chain variable region gene downstream index primer, and the rat antibody heavy chain variable region gene upstream primer is 15 single-stranded DNA (deoxyribonucleic acid) with nucleotide sequences of 17-31 in a sequence table respectively; the downstream index primer of the rat heavy chain variable region gene is single-stranded DNA with a nucleotide sequence of 65 in a sequence table; the light chain variable region gene primer composition consists of a rat light chain variable region gene upstream primer and a rat light chain variable region gene downstream index primer, wherein the rat light chain variable region gene upstream primer is 15 single-stranded DNA (deoxyribonucleic acid) with nucleotide sequences of sequence 1-sequence 15 in a sequence table respectively; the rat light chain variable region gene downstream index primer is single-stranded DNA of a sequence 64 in a sequence table;
B. The hybridoma is a mouse hybridoma, the heavy chain variable region gene primer composition consists of a mouse antibody heavy chain variable region gene upstream primer and a mouse heavy chain variable region gene downstream index primer, and the mouse antibody heavy chain variable region gene upstream primer is 15 single-stranded DNA (deoxyribonucleic acid) with nucleotide sequences of 46-60 in a sequence table respectively; the downstream index primer of the mouse heavy chain variable region gene is single-stranded DNA with a nucleotide sequence of sequence 63 in a sequence table; the light chain variable region gene primer composition consists of a mouse light chain variable region gene upstream primer and a mouse light chain variable region gene downstream index primer, wherein the mouse light chain variable region gene upstream primer is 12 single-stranded DNA (deoxyribonucleic acid) with nucleotide sequences of 33-44 in a sequence table respectively; the mouse light chain variable region gene downstream index primer is single-stranded DNA of a sequence 62 in a sequence table.
5. The composition for amplifying the monoclonal antibody variable region gene is characterized by comprising a heavy chain variable region gene primer composition and a light chain variable region gene primer composition, wherein the heavy chain variable region gene primer composition comprises a heavy chain variable region gene upstream primer and a heavy chain variable region gene downstream index primer, the heavy chain variable region gene downstream index primer comprises N primers, the 5' end of each primer contains a Barcode sequence, the other heavy chain variable region gene specific sections are different from each other, and the Barcode sequences of the heavy chain variable region gene downstream index primers are different from each other;
The light chain variable region gene primer composition consists of a light chain variable region gene upstream primer and a light chain variable region gene downstream index primer, wherein the light chain variable region gene downstream index primer consists of N primers, the 5' end of each primer contains the Barcode sequence, the rest is a light chain variable region gene specific section, and the Barcode sequence of each light chain variable region gene downstream index primer is different;
the barcode sequence is single-stranded DNA for distinguishing different hybridoma cells, and the barcode sequence is any one of 68 single-stranded DNA molecules: b1, 5'-TCGTCATG-3'; b2, 5'-GTCAGTCC-3'; b3, 5'-CGATTACG-3'; b4, 5'-TCTACGAG-3'; b5, 5'-TAACGTGC-3'; b6, 5'-CGAAGGTT-3'; b7, 5'-CACGCATT-3'; b8, 5'-TACCAGGT-3'; b9, 5'-TTACCGAG-3'; b10, 5 '-AGTCACT-3'; b11, 5'-TCGCATTC-3'; b12, 5'-CATGGTAC-3'; b13, 5 '-CACGGGACT-3'; b14, 5'-CTGTAGCA-3'; b15, 5'-AGAGCTTG-3'; b16, 5'-AGCATAGC-3'; b17, 5 '-CCTAACAT-3'; b18, 5'-GTCGTTGA-3'; b19, 5'-TGCGTATC-3'; b20, 5 '-AGCTACC-3'; b21, 5'-GAACGGTA-3'; b22, 5'-GGATTGTC-3'; b23, 5'-TACCGTCT-3'; b24, 5'-CGAGTATC-3'; b25, 5'-TGTACGCT-3'; b26, 5'-TCCTCTGA-3'; b27, 5'-AGCGTATC-3'; b28, 5 '-TCACC-3'; b29, 5'-GACAGTAC-3'; b30, 5 '-GTCCTTC-3'; b31, 5'-GGAGCAAT-3'; b32, 5'-AGGAACAG-3'; b33, 5'-CAGTGCTA-3'; b34, 5 '-CTAGCTAGTAG-3'; b35, 5'-TGCAAGCA-3'; b36, 5'-CGCATCTA-3'; b37, 5'-CGTATCTG-3'; b38, 5'-GTAGACCT-3'; b39, 5'-CGCCATTA-3'; b40, 5'-ATGACACC-3'; b41, 5'-CAAGTTGA-3'; b42, 5'-TGCTGAAG-3'; b43, 5'-ATGACCGT-3'; b44, 5'-TGGCGAGA-3'; b45, 5'-GCACTTCC-3'; b46, 5'-GTTACCGA-3'; b47, 5'-GACGTCAC-3'; b48, 5'-GTTACAGC-3'; b49, 5'-TCGATTGC-3'; b50, 5'-GACGATCT-3'; b51, 5'-TGACGCTA-3'; b52, 5'-ATAACGCG-3'; b53, 5'-GATTCACC-3'; b54, 5'-GGCAAGTA-3'; b55, 5'-GATCTAGC-3'; b56, 5'-CATTGCGA-3'; b57, 5'-GCTGTACA-3'; b58, 5'-TAGCAGCT-3'; b59, 5'-AGCGACAT-3'; b60, 5'-CATTAGCC-3'; b61, 5 '-catctgca-3'; b62, 5'-GTCATCGT-3'; b63, 5'-GGACTTCA-3'; b64, 5'-CAGGATCA-3'; b65, 5'-GAACGATG-3'; b66, 5'-CGACTTGC-3'; b67, 5'-GATTCCGT-3' and B68, 5'-CCGCTTAC-3'.
6. The composition of claim 5, wherein the composition is a or B:
A. the monoclonal antibody is produced by a rat hybridoma, the heavy chain variable region gene primer composition consists of an upstream primer of a rat antibody heavy chain variable region gene and a downstream index primer of the rat heavy chain variable region gene, and the upstream primer of the rat antibody heavy chain variable region gene is 15 single-stranded DNA (deoxyribonucleic acid) with nucleotide sequences of 17-31 in a sequence table respectively; the downstream index primer of the rat heavy chain variable region gene is single-stranded DNA with a nucleotide sequence of 65 in a sequence table; the light chain variable region gene primer composition consists of a rat light chain variable region gene upstream primer and a rat light chain variable region gene downstream index primer, wherein the rat light chain variable region gene upstream primer is 15 single-stranded DNA (deoxyribonucleic acid) with nucleotide sequences of sequence 1-sequence 15 in a sequence table respectively; the rat light chain variable region gene downstream index primer is single-stranded DNA of a sequence 64 in a sequence table;
B. the monoclonal antibody is produced by a mouse hybridoma, the heavy chain variable region gene primer composition consists of an upstream primer of a mouse antibody heavy chain variable region gene and a downstream index primer of the mouse heavy chain variable region gene, and the upstream primer of the mouse antibody heavy chain variable region gene is 15 single-stranded DNA (deoxyribonucleic acid) with nucleotide sequences of 46-60 in a sequence table respectively; the downstream index primer of the mouse heavy chain variable region gene is single-stranded DNA with a nucleotide sequence of sequence 63 in a sequence table; the light chain variable region gene primer composition consists of a mouse light chain variable region gene upstream primer and a mouse light chain variable region gene downstream index primer, wherein the mouse light chain variable region gene upstream primer is 12 single-stranded DNA (deoxyribonucleic acid) with nucleotide sequences of 33-44 in a sequence table respectively; the mouse light chain variable region gene downstream index primer is single-stranded DNA of a sequence 62 in a sequence table.
7. A kit for amplifying an antibody variable region gene, comprising the composition of claim 5 or 6.
8. Use of the method of claim 1 for high throughput sequencing of monoclonal antibody variable region genes.
9. Use of the composition of claim 5 or 6 in high throughput sequencing of monoclonal antibody variable region genes.
10. An apparatus for constructing an antibody treeing comprising:
a PCR amplification device for amplifying variable region genes of monoclonal antibodies secreted by N hybridoma cells using the composition of claim 5 or 6 to obtain antibody variable region gene products; the N is a natural number greater than or equal to 2;
the sequencing device is used for sequencing the gene product comprising the antibody variable region to obtain a sequencing result;
and the analysis device is used for constructing a evolutionary tree of the monoclonal antibody based on the sequencing result.
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