CN117004700A - Method for high-throughput sequencing of monoclonal antibody variable region genes, composition and kit used by method - Google Patents

Abstract

The invention discloses a method for high-throughput sequencing of monoclonal antibody variable region genes in the field of nucleic acid determination and detection, as well as compositions and kits used therein. The technical problem to be solved by the present invention is how to improve the sequencing efficiency of antibody variable regions. The method for high-throughput sequencing of monoclonal antibody variable region genes of the present invention includes: 1) obtaining cDNA of hybridoma cells secreting monoclonal antibodies; 2) using cDNA as a template and using primer compositions to perform PCR amplification to obtain PCR Product; the primer composition consists of a heavy chain primer (including a heavy chain upstream primer and a heavy chain downstream primer) and a light chain primer (including a light chain upstream primer and a light chain downstream primer); 5 of the heavy chain downstream primer and the light chain downstream primer Both ends contain barcode sequences; 3) Mix the PCR products for sequencing; perform data analysis on the sequencing results to obtain the sequence of the monoclonal antibody variable region gene.

Description

Methods for high-throughput sequencing of monoclonal antibody variable region genes and compositions and Reagent test kit

Technical field

The present invention relates to methods for high-throughput sequencing of monoclonal antibody variable region genes in the field of nucleic acid determination and detection, as well as compositions and kits therefor.

Background technique

Hybridoma cell technology is the most widely used and mature antibody preparation technology platform. Hybridoma cells are cells formed by the fusion of mouse myeloma cells and B lymphocytes. It has the characteristics of unlimited reproduction of myeloma cells and the ability of lymphocytes to secrete specific antibodies. Although hybridoma cells have the characteristics of immortalization, 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, it is constructed into an expression vector and the corresponding antibody can be obtained through recombinant expression. Therefore, it is very important to obtain the antibody variable region sequence through sequencing, which allows the antibody to be preserved in the form of base sequence.

Existing antibody variable region sequencing uses first-generation sequencing technology. First-generation sequencing is a DNA end termination sequencing technology invented by Sanger et al. in the mid-1970s. An extension reaction occurs under the action of DNA polymerase, template, radioactive isotope-labeled primer, dNTP and ddNTP. Due to the presence of ddNTPs, DNA extension fragments of different lengths will be formed. Slab gel electrophoresis is then used to separate the products of the four reactions using four electrophoresis lanes, and the corresponding DNA sequences can be read out in sequence. The advantages of the first-generation Sanger sequencing technology are: long sequencing reads, short sequencing time, and high accuracy.

The antibody sequencing process first extracts hybridoma cell RNA and reverse-transcribes it into cDNA. Using cDNA as a template, specific primers are used to amplify the antibody variable region sequence, and the variable region sequence is sequenced for one generation. Although the application of first-generation sequencing technology can meet the needs of antibody sequencing, each sequencing reaction can only complete the sequencing of one antibody light chain or heavy chain variable region. If the sequencing of the amplified product is unsuccessful (for example, there are overlapping peaks in the sequencing results; the signal is weak, Product sequencing fails), TA cloning is also required. Therefore, the application of existing sequencing methods not only has high sequencing costs and low throughput, but is also time-consuming and labor-intensive, and cannot meet the needs of high-throughput sequencing of antibody variable regions.

With the development of second-generation sequencing technology, sequencing projects have greatly reduced sequencing costs while also greatly increasing sequencing speed and maintaining high accuracy. Although second-generation sequencing is lower than first-generation sequencing in terms of sequence read length, It can fully meet the requirements of antibody sequencing. Based on the innovation of sequencing technology and the demand for antibody sequencing throughput, there is an urgent need to develop high-throughput antibody sequencing methods based on second-generation sequencing.

Contents of the invention

The technical problem to be solved by the present invention is how to improve the sequencing efficiency of antibody variable region genes and reduce sequencing costs.

In order to solve the above technical problems, the present invention provides a method for high-throughput sequencing of monoclonal antibody variable region genes.

The method for high-throughput sequencing of monoclonal antibody variable region genes provided by the invention includes the following steps:

1) Obtain cDNA of N hybridoma cells, each of the N hybridoma cells secretes a monoclonal antibody, and N is a natural number greater than or equal to 2;

2) Using the cDNA of each hybridoma cell as a template, use the heavy chain variable region gene primer composition and the light chain variable region gene primer composition to perform PCR to amplify the monoclonal antibodies secreted by each hybridoma cell. The heavy chain variable region gene and the light chain variable region gene were obtained to obtain the heavy chain variable region gene PCR product of the monoclonal antibody secreted by each hybridoma cell and the light chain of the monoclonal antibody secreted by each hybridoma cell. Variable region gene PCR products; mix and sequence the heavy chain variable region gene PCR products of monoclonal antibodies secreted by N hybridoma cells to obtain the heavy chain variable region gene PCR products of monoclonal antibodies secreted by N hybridoma cells Sequencing results; the PCR products of the light chain variable region genes of the monoclonal antibodies secreted by the N hybridoma cells are mixed and sequenced to obtain the sequencing results 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 is composed 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 is composed of N types of primers, each primer The 5' ends of all contain Barcode sequences (sequence-specific tags), and the rest are heavy chain variable region gene-specific segments. The Barcode sequences of the downstream index primers of each heavy chain variable region gene are different;

The light chain variable region gene primer composition is composed of a light chain variable region gene upstream primer and a light chain variable region gene downstream index primer. The light chain variable region gene downstream index primer is composed of N types of primers, each primer The 5' ends of all contain Barcode sequences (sequence-specific tags), and the rest are light chain variable region gene-specific segments. The Barcode sequences of the downstream index primers of each light chain variable region gene are different;

3) Sequencing the heavy chain variable region gene PCR product of the monoclonal antibody secreted by the N hybridoma cells obtained in 2) and the light chain variable region gene PCR product of the monoclonal antibody secreted by the N hybridoma cells The sequencing results were analyzed and the sequences of the monoclonal antibody variable region genes secreted by each hybridoma cell were obtained.

In this method, the Barcode sequence is a single-stranded DNA used to distinguish the antibody heavy chain or light chain of different hybridoma cells. The Barcode sequence may be a single-stranded DNA consisting of 8 nucleotides.

The Barcode sequence can be specifically 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'-CACGCAT-3'; B8, 5'-TACCAGGT-3'; B9, 5'-TTACCGAG-3'; B10, 5'-AGTCACCT-3'; B11, 5'-TCGCATTC-3'; B12, 5'-CATGGTAC-3'; B13, 5'-CACGGACT-3'; B14, 5'-CTGTAGCA-3'; B15, 5'-AGAGCTTG-3'; B16, 5'-AGCATAGC-3'; B17, 5'-CCTACCAT-3'; B18, 5'-GTCGTTGA-3'; B19, 5'-TGCGTATC-3'; B20, 5'-AGCTAACC-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'-TCATCACC-3'; B29, 5'-GACAGTAC-3'; B30, 5'-GTCCTTAC-3'; B31, 5'-GGAGCAAT-3'; B32, 5'-AGGAACAG-3'; B33, 5'-CAGTGCTA-3'; B34, 5'-CTAGCTAG-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'-CATCTCCA-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 present invention, the heavy chain variable region gene upstream primer is a single-stranded DNA fragment that specifically binds to the heavy chain signal peptide encoding gene of the monoclonal antibody, and the heavy chain variable region gene downstream index primer is composed of the A single-stranded DNA fragment formed by connecting the barcode sequence and a heavy chain variable region gene-specific segment. The heavy chain variable region gene-specific segment specifically binds to the heavy chain signal peptide region and constant of the monoclonal antibody. region of DNA.

In the present invention, the light chain variable region gene upstream primer is a single-stranded DNA fragment that specifically binds to the light chain signal peptide encoding gene of the monoclonal antibody, and the light chain variable region gene downstream index primer is composed of A single-stranded DNA fragment formed by connecting the barcode sequence and the light chain variable region gene-specific segment. The light chain variable region gene-specific segment specifically binds to the light chain signal peptide region and constant of the monoclonal antibody. region of DNA.

In the above method, the hybridoma may be a rat hybridoma or a mouse hybridoma.

The rat hybridoma cells can secrete rat antibodies.

The mouse hybridoma cells can secrete mouse antibodies.

In the above method, in a specific embodiment, the primer composition can be A or B:

A. The hybridoma is a rat hybridoma, and 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. Composition, the upstream primer of the rat heavy chain variable region gene is 15 kinds of single-stranded DNA whose nucleotide sequences are sequence 17-sequence 31 in the sequence list, and the downstream index primer of the rat heavy chain variable region gene The nucleotide sequence is the single-stranded DNA of sequence 65 in the sequence list; the rat antibody light chain variable region gene primer composition consists of the rat light chain variable region gene upstream primer and the rat light chain variable region gene The downstream index primer consists of 15 kinds of single-stranded DNA whose nucleotide sequences are sequence 1 to sequence 15 in the sequence list. The upstream primers of the rat light chain variable region gene are 15 kinds of single-stranded DNA. The index primer is the single-stranded DNA of sequence 64 in the sequence list;

B. The hybridoma is a mouse hybridoma, and 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. Composition, the mouse antibody heavy chain variable region gene upstream primer is 15 kinds of single-stranded DNA whose nucleotide sequences are sequence 46-sequence 60 in the sequence list, and the mouse heavy chain variable region gene downstream index primer The nucleotide sequence is the single-stranded DNA of sequence 63 in the sequence list; 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 The downstream index primer consists of 12 single-stranded DNAs whose nucleotide sequences are sequence 33-sequence 44 in the sequence list, and the mouse light chain variable region gene downstream primers are The index primer is the single-stranded DNA of sequence 62 in the sequence list.

The present invention also provides a composition for amplifying monoclonal antibody variable region genes. The composition is composed of a heavy chain variable region gene primer composition and a light chain variable region gene primer composition. The heavy chain variable region gene primer composition The 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, and the 5' end of each primer contains Barcode sequence (sequence-specific tag), and the rest are heavy chain variable region gene-specific segments. The Barcode sequences of the downstream index primers of each heavy chain variable region gene are different; the light chain variable region gene primer composition is composed of The light chain variable region gene upstream primer and the light chain variable region gene downstream index primer are composed of N types of primers, and the 5' end of each primer contains the Barcode sequence (sequence Specific tag), and the rest are specific segments of the light chain variable region gene. The Barcode sequences of the downstream index primers of each light chain variable region gene are different.

In the composition, the barcode sequence is a single-stranded DNA used to distinguish different hybridoma cells, and the barcode sequence is any one of the following 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'-AGTCACCT-3'; B11, 5'-TCGCATTC-3' ;B12, 5'-CATGGTAC-3'; B13, 5'-CACGGACT-3'; B14, 5'-CTGTAGCA-3'; B15, 5'-AGAGCTTG-3'; B16, 5'-AGCATAGC-3' ;B17, 5'-CCTACCAT-3'; B18, 5'-GTCGTTGA-3'; B19, 5'-TGCGTATC-3'; B20, 5'-AGCTAACC-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'-TCATCACC-3'; B29, 5'-GACAGTAC-3'; B30, 5'-GTCCTTAC-3'; B31, 5'-GGAGCAAT-3' ;B32, 5'-AGGAACAG-3'; B33, 5'-CAGTGCTA-3'; B34, 5'-CTAGCTAG-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'-CATCTCCA-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'.

Among the compositions for amplifying monoclonal antibody variable region genes described above, the composition may be A or B:

A. The monoclonal antibody is produced by a rat hybridoma, and the heavy chain variable region gene primer composition is composed of a rat antibody heavy chain variable region gene upstream primer and a rat heavy chain variable region gene downstream index primer. , the upstream primers of the rat antibody heavy chain variable region gene are 15 single-stranded DNAs whose nucleotide sequences are sequence 17-sequence 31 in the sequence list, and the downstream index primers of the rat heavy chain variable region gene are The nucleotide sequence is the single-stranded DNA of sequence 65 in the sequence list; 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. , the upstream primers of the rat light chain variable region gene are 15 single-stranded DNAs whose nucleotide sequences are sequence 1 to sequence 15 in the sequence list, and the downstream index primers of the rat light chain variable region gene are sequence Single-stranded DNA of sequence 64 in the list;

B. The monoclonal antibody is produced by a mouse hybridoma, and 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. , the mouse antibody heavy chain variable region gene upstream primers are 15 kinds of single-stranded DNA whose nucleotide sequences are sequence 46-sequence 60 in the sequence list; the mouse heavy chain variable region gene downstream index primer is The nucleotide sequence is the single-stranded DNA of sequence 63 in the sequence list; the light chain variable region gene primer composition is composed of a mouse light chain variable region gene upstream primer and a mouse light chain variable region gene downstream index primer. , the mouse light chain variable region gene upstream primers are 12 single-stranded DNAs whose nucleotide sequences are sequence 33-sequence 44 in the sequence list; the mouse light chain variable region gene downstream index primers are sequence Single-stranded DNA of sequence 62 in the list.

The present invention also provides a kit for amplifying the variable region of an antibody, which contains the primer composition described above.

The present invention also provides the application of the method described above in high-throughput sequencing of monoclonal antibody variable region genes.

The present invention also provides the application of the composition described above in high-throughput sequencing of monoclonal antibody variable region genes.

The present invention also provides the application of the aforementioned kit in preparing high-throughput sequencing products of monoclonal antibody variable region genes.

The invention also provides a device for constructing an antibody evolutionary tree, including:

PCR amplification device, the amplification device is used to amplify the variable region genes of monoclonal antibodies secreted by N hybridoma cells using the aforementioned composition to obtain an antibody variable region gene amplification product; N is a natural number greater than or equal to 2;

A sequencing device, the sequencing device is used to sequence gene products including the antibody variable region and obtain sequencing results;

An analysis device, the analysis device is used to construct an evolutionary tree of the monoclonal antibody based on the sequencing results.

In the present invention, the sequencing method may be second-generation sequencing (such as Illumina-Hiseq method).

The present invention adds a unique label to the amplification product of each antibody light chain and heavy chain variable region by adding different barcode sequences (different linkers) to the downstream primers of the antibody variable region, and then adds the light chain and heavy chain variable region amplification products of N antibodies. The amplified products of heavy chain and heavy chain variable regions were mixed, and the mixed amplified products were subjected to next-generation sequencing (Illumina-Hiseq). The mixed product sequence sequencing results are analyzed using the company's independently developed antibody sequence analysis software. Not only can hundreds of antibody variable region sequences be obtained in one sequencing, but it also solves the problems often encountered in first-generation sequencing of variable region products (sanger method). Problems such as overlapping peaks and sequencing failure due to low product concentration eliminate the need for TA cloning. Therefore, a single next-generation sequencing (NGS) can obtain hundreds of antibody light chain and heavy chain variable region sequences. The sequencing results obtained by the sequencing method of the present invention are simple to analyze, easy to operate, and highly accurate. There is no need to perform TA cloning of problematic results. Compared with traditional sequencing methods, it not only saves manpower and time, but also reduces sequencing costs.

Description of the drawings

Figure 1 shows the electrophoresis results after RNA extraction with different antibodies. Lanes A1-A5 are rat antibodies, and lanes A6-A34 are mouse antibodies.

Figure 2 is a flow chart of the antibody sequence analysis program.

Figure 3 shows the amplification results of light chain variable region genes of different antibodies using primer combinations containing different barcodes. Lanes A1-A5 are rat antibodies, and lanes A6-A34 are mouse antibodies.

Figure 4 shows the amplification results of heavy chain variable region genes of different antibodies using primer combinations containing different barcodes. Lanes A1-A5 are rat antibodies, and lanes A6-A34 are mouse antibodies.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions.

Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Quantitative experiments in the following examples were repeated three times unless otherwise specified.

The DNA polymerase in the present invention is 2×TransTaq® High Fidelity (HiFi) PCR SuperMix II(-dye) (Beijing Quanshijin Biotechnology Co., Ltd., AS131-21). Reagents for electrophoresis detection and product recovery: agarose (Beijing Quan-Gold Biotechnology Co., Ltd., GS201-01); nucleic acid dye (Qual-gold, GS101-01); DNA loading buffer (Qual-gold, GH101-01); DNA marker (full gold, Trans2K® DNA Marker); EasyPure®Quick Gel Extraction Kit (full gold, EG101-01); animal tissue/cell total RNA extraction kit (Tiangen, DP451); cell culture medium: DMEM cells Medium (Thermofisher, 11971025); serum: Australian fetal calf serum (special grade) (Minhai, SA102.01); double antibody: Penicillin-Streptomycin (Thermo, 15070063); cell culture flask T25 (Thermo, 15070063).

In the nucleotide sequence of this article, 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 , N is A, T, C or G.

Example 1. Establishment of high-throughput sequencing method for rat and mouse antibody variable region genes

1. Extraction of cellular RNA

Thirty-four types of hybridoma cells, named hybridoma cells A1-A34, were cultured. Each of the 34 types of hybridoma cells secreted a monoclonal antibody, whose names were antibodies A1-A34. Among them, hybridoma cells A1-A5 are rat hybridoma cells, and hybridoma cells A6-A34 are mouse hybridoma cells. The animal cell culture method is as follows: Use a T25 cell culture bottle to culture hybridoma cells in culture medium (1×DMEM+10% fetal bovine serum+1% double antibody), place the cell culture bottle in a carbon dioxide incubator, and culture conditions: 37℃, 5% CO ₂ . When the cells grow to about 5×10 ⁶ -1×10 ⁷ /flask, collect the cells.

Use an RNA extraction kit to extract RNA from rat and mouse hybridoma cells, conduct electrophoresis verification of the extracted RNA, and run gel to observe the integrity of the RNA. The results are shown in Figure 1. There are two clear rRNA bands of 28S and 18S in the extracted total RNA, and the intensity of the 28S rRNA band is about twice that of the 18S rRNA band, indicating that the RNA integrity is good.

SMARTer RACE 5’/3’Kit kit (Clontech, Cat. No. 634499) was used to obtain the cDNA of hybridoma cells by reverse transcription, and the cDNA of hybridoma cells A1-A34 were obtained respectively.

2. Synthesis of light chain variable region gene primers and heavy chain variable region gene primers for rat and mouse antibodies

1) There are 15 types of upstream primers for rat antibody light chain variable region genes, as follows:

(1) RATKF1: 5’-RAATTGTSHTGACYCAGTCTC-3’ (sequence 1);

(2) RATKF2: 5’-GAAACTGTGATGACCCAGTC-3’ (sequence 2);

(3) RATKF3: 5’-CAGGCTGTTGTGACTCAGG-3’ (sequence 3);

(4) RATKF4: 5’-RACRTCCAGWTRACCCAGWCT-3’ (sequence 4);

(5) RATKF5: 5’-GACATCCAYATGACWCAGWM-3’ (sequence 5);

(6) RATKF6: 5’-GACATYSRGATGACYMAGTCTC-3’ (sequence 6);

(7) RATKF7: 5’-GACATTKYGATRACCCARTMT-3’ (sequence 7);

(8) RATKF8: 5’-GATATTGTGATGACHCARRS-3’ (sequence 8);

(9) RATKF9: 5’-GATGTTGTTTTTGGTGACACA-3’ (sequence 9);

(10) RATKF10: 5’-GATGTTRTGMTGRCCCAGAC-3’ (sequence 10);

(11) RATKF11: 5’-GAMATTRTRCTVACCCAGTCT-3’ (sequence 11);

(12) RATKF12: 5’-GAWAWTGTKCTMMCTCAGTC-3’ (sequence 12);

(13) RATKF13: 5’-GACGTTGTGCTGACTCAGTC-3’ (sequence 13);

(14) RATKF14: 5’-CAGCCCGTGCTGCATCAG-3’ (sequence 14);

(15) RATKF15: 5’-CAGWTCACGCTSACYCARCM-3’ (sequence 15).

The downstream primer of the rat antibody light chain variable region gene is the index primer, and its general section (specific section of the rat antibody light chain variable region gene) is RATKR, as follows: RATKR: 5'-ACGTTTYATTTCCARCTKSGTC-3' (sequence 16).

There are 15 types of upstream primers for rat antibody heavy chain variable region genes, 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’ (sequence 22);

(7) RATHF7: 5’-GATCAGGTCCAGYTGCAGCARTS-3’ (sequence 23);

(8) RATHF8: 5’-GATCAGATTCAGCTGCARCAGTG-3’ (sequence 24);

(9) RATHF9: 5’-GATCAAACAGTCCAGCTACAGCAGTC-3’ (sequence 25);

(10) RATHF10: 5’-GATCAAGARGTCCWGCTGCAKCAGTM-3’ (sequence 26);

(11) RATHF11: 5’-GATCAGGTTMMCTGMAASAGTC-3’ (sequence 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’ (sequence 31).

The downstream primer of the rat antibody heavy chain variable region gene is the index primer, and its general section (specific section of the rat antibody heavy chain variable region gene) is RATHR, specifically as follows: RATHR: 5'-GATCKGAGGASACGGTGACCRKGG-3' (sequence 32).

There are 12 types of upstream primers for mouse antibody light chain variable region genes, as follows:

(1) MKF1: 5’-GCTTACAGGTGCCAGATGT-3’ (sequence 33);

(2) MKF2: 5’-TCAATTGTAGRTGCCAGATGT-3’ (sequence 34);

(3) MKF3: 5’-TTACAGTAGGTGTCAGATGT-3’ (sequence 35);

(4) MKF4: 5’-CAGTCGTAGTTGTCAGATGT-3’ (sequence 36);

(5) MKF5: 5’-CCTCCTCTTGGCCAAGA-3’ (sequence 37);

(6) MKF6: 5’-TTATATGGAGCTGATGGG-3’ (sequence 38);

(7) MKF7: 5’-GTGTCTGGTGCTCATGGG-3’ (sequence 39);

(8) MKF8: 5’-CTSTGGTTGTCTGGTGTTGA-3’ (sequence 40);

(9) MKF9: 5’-GTCTCTGATTCTAGGGCA-3’ (sequence 41);

(10) MKF10: 5’-TGCTKCKCTGGGTTCCAG-3’ (sequence 42);

(11) MKF11: 5’-TTGCAGGTGTTGACGGA-3’ (sequence 43);

(12) MKF12: 5’-GTAGGTGCCTCGTGCAC-3’ (sequence 44).

The downstream primer of the mouse antibody light chain variable region gene is the index primer, and its universal segment (specific segment of the mouse antibody light chain variable region gene) is MKR, specifically as follows: MKR: 5'-CGACTAGTCGACTGGTGGGAAGATGGATACAG-3' (sequence 45).

There are 15 types of upstream primers for mouse antibody heavy chain variable region genes, as follows:

(1) MHF1:5’-ACTGCAGGTRTCCACTCC-3’ (sequence 46);

(2) MHF2:5’-RCTACAGGTGTCCACTCC-3’ (sequence 47);

(3) MHF3:5’-ACTGCAGGTGTCCWMTCC-3’ (sequence 48);

(4) MHF4:5’-RCTRCAGGTGTKCACTCC-3’ (sequence 49);

(5) MHF5:5’-GCTACAGGTGCTCACTCC-3’ (sequence 50);

(6) MHF6:5’-AYTGCAGGTGTCCAYTGC-3’ (sequence 51);

(7) MHF7:5’-GCTAMMGGTGTCCACTTC-3’ (sequence 52);

(8) MHF8:5’-RCTRCAGGYGTCCACTCT-3’ (sequence 53);

(9) MHF9:5’-CCAAGCTGTATCCTTTCC-3’ (sequence 54);

(10) MHF10:5’-CCAAGCTGTGTCCTRTCC-3’ (sequence 55);

(11) MHF11:5’-GTTTTAAAAGGTGTCCTGTG-3’ (sequence 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’ (sequence 60).

The downstream primer of the mouse antibody heavy chain variable region gene is the index primer, and its general section (specific section of the mouse antibody heavy chain variable region gene) is MHR, as follows: MHR:5'-GGATAGACWGATGGGGSTGTYGTT-3' (sequence 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 index primer downstream of the light chain variable region gene both contain Barcode sequences, and the rest are universal segments. Barcode sequences are single-stranded DNA used to distinguish between different antibody heavy or light chains. In this embodiment, the Barcode sequence is a single-stranded DNA composed of 8 nucleotides.

1) Barcode sequence synthesis

The present invention provides 68 kinds of Barcode sequences of B1-B68, specifically 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'-CACGCAT-3'; B8, 5'-TACCAGGT-3'; B9, 5'-TTACCGAG-3'; B10, 5'-AGTCACCT-3'; B11, 5'-TCGCATTC-3'; B12, 5'-CATGGTAC-3'; B13, 5'-CACGGACT-3'; B14, 5'-CTGTAGCA-3'; B15, 5'-AGAGCTTG-3'; B16, 5'-AGCATAGC-3'; B17, 5'-CCTACCAT-3'; B18, 5'-GTCGTTGA-3'; B19, 5'-TGCGTATC-3'; B20, 5'-AGCTAACC-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'-TCATCACC-3'; B29, 5'-GACAGTAC-3'; B30, 5'-GTCCTTAC-3'; B31, 5'-GGAGCAAT-3'; B32, 5'-AGGAACAG-3'; B33, 5'-CAGTGCTA-3'; B34, 5'-CTAGCTAG-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'-CATCTCCA-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', by Generic Biosynthesis.

2) Index primer synthesis

Synthesize the index primer by adding the barcode sequence in 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 mark an antibody light chain or heavy chain, that is, there is a one-to-one correspondence between a barcode sequence and an antibody light chain or heavy chain variable region gene sequence. Step 1) 68 kinds of Barcodes are provided, and the index primer design is as follows:

A. Index primer downstream of mouse antibody light chain variable region gene:

Add the barcode sequence to the 5' end of the universal segment MKR of the downstream primer of the mouse antibody light chain to synthesize the downstream index primer of the mouse antibody light chain variable region gene: 5'-NNNNNNNNCGACTAGTCGACTGGTGGGAAGATGGATACAG-3' (sequence 62 in the sequence listing). Positions 1-8 (5’-NNNNNNNN-3’) of sequence 62 are Barcodes. For the nucleotide sequences of the 68 Barcodes, see step 1) above. This step uses a total of 29 different barcode sequences from B1 to B29 in step 1) above to obtain 29 mouse antibody light chain variable region gene downstream index primers.

B. Index primer downstream of mouse antibody heavy chain variable region gene:

Add the barcode sequence to the 5' end of the universal segment MHR of the downstream primer of the mouse antibody heavy chain to synthesize the downstream index primer of the mouse antibody heavy chain variable region gene: 5'-NNNNNNNNGGATAGACWGATGGGGSTGTYGTT-3' (sequence 63 in the sequence listing). Positions 1-8 (5'-NNNNNNNN-3') of sequence 63 are Barcodes. This step uses a total of 29 different barcode sequences from B30-B58 in step 1) above to obtain 29 mouse antibodies containing different barcode sequences. Index primer downstream of the heavy chain variable region gene.

C. Index primer downstream of rat antibody light chain variable region gene:

Add the barcode sequence to the 5' end of the universal segment RATKR of the downstream primer of the rat antibody light chain to synthesize the downstream index primer of the rat antibody light chain variable region gene: 5'-NNNNNNNNACGTTTYATTTCCARCTKSGTC-3' (sequence 64 in the sequence listing). Positions 1-8 (5’-NNNNNNNN-3’) of sequence 64 are Barcodes. For the nucleotide sequences of the 68 Barcodes, see step 1) above. This step uses a total of 5 different barcode sequences from B59-B63 in step 1) above to obtain 5 downstream index primers for rat antibody light chain variable region genes containing different barcode sequences.

D. Index primer downstream of rat antibody heavy chain variable region gene:

Add the barcode sequence to the 5' end of the universal section RATHR of the downstream primer of the rat antibody heavy chain to synthesize the downstream index primer of the rat antibody heavy chain variable region gene: 5'-NNNNNNNNGATCKGAGGASACGGTGACCRKGG-3' (sequence 65 in the sequence listing). Positions 1-8 (5’-NNNNNNNN-3’) of sequence 65 are Barcodes. For the nucleotide sequences of the 68 Barcodes, see step 1) above. This step uses a total of 5 different barcode sequences from B64 to B68 in step 1) above to obtain 5 downstream index primers for rat antibody heavy chain variable region genes containing different barcode sequences.

4. Establishment of high-throughput sequencing methods for rat and mouse antibody variable regions

1) Mixing of amplification primers

Before dissolving the primers, use a centrifuge to centrifuge the primer powder obtained in steps 2 and 3 at 12,000 rpm for 10 minutes. Use enzyme-free water for dissolution.

Mix a total of 15 types of upstream primers for the rat antibody light chain variable region gene in step 2 to obtain an LR mix. In the LR mix, the content of each type of upstream primer for the rat antibody light chain variable region gene is 10 μmol/L.

Mix a total of 15 types of upstream primers for the rat antibody heavy chain variable region gene in Step 2 to obtain an HR mix. In the HR mix, the content of each upstream primer for the rat antibody heavy chain variable region gene is 10 μmol/L.

Mix a total of 12 types of mouse antibody light chain variable region gene upstream primers from step 2 to obtain LM mix. In the LM mix, the content of each mouse antibody light chain variable region gene upstream primer is 10 μmol/L.

Mix a total of 15 types of mouse antibody heavy chain variable region gene upstream primers from step 2 to obtain HM mix. In the HM mix, the content of each mouse antibody heavy chain variable region gene upstream primer is 10 μmol/L.

2) PCR amplification reaction

A. Antibody heavy chain variable region gene PCR system:

The only difference between the rat antibody heavy chain variable region gene PCR system and the mouse antibody heavy chain variable region PCR system is that the cDNA template, upstream primer and downstream index primer are different, and the rest are the same.

The rat antibody heavy chain variable region gene PCR system has a total of 5 PCR systems. These 5 PCR systems are named A1 rat antibody heavy chain variable region gene PCR system and A2 rat antibody heavy chain variable region gene PCR system. , A3 rat antibody heavy chain variable region gene PCR system, A4 rat antibody heavy chain variable region gene PCR system and A5 rat antibody heavy chain variable region gene PCR system. The upstream primers of these five PCR systems are all HR mix; the cDNA and downstream index primers of these five PCR systems are all different, among which: the cDNA in the A1 rat antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A1 , the Barcode sequence contained in the downstream index primer is B1, 5'-TCGTCATG-3', the cDNA in the A2 rat antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A2, and the Barcode sequence contained in the downstream index primer is B2 , 5'-GTCAGTCC-3', A3 The cDNA in the rat antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A3, and the Barcode sequence contained in the downstream index primer is B3, 5'-CGATTACG-3', A4 The cDNA in the rat antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A4. The Barcode sequence contained in the downstream index primer is B4, 5'-TCTACGAG-3', and the A5 rat antibody heavy chain variable region PCR system The cDNA in is the cDNA of hybridoma cell A5, and the Barcode sequence contained in the downstream index primer is B5, 5'-TAACGTGC-3'.

Rat antibody heavy chain variable region gene PCR system: 1 μL of cDNA from one of the A1-A5 hybridoma cells in step 1, 1 μL of rat antibody heavy chain upstream primer (HR mix), and downstream index primer (containing The specific Barcode sequence corresponds to the cDNA of different hybridoma cells one-to-one) 1 μL, 2×Trans Taq HiFi PCR Super Mix II 5 μL, ddH ₂ O 2 μL, the total volume is 10 μL.

The mouse antibody heavy chain variable region gene PCR system has a total of 29 PCR systems. These 5 PCR systems are named A6 mouse antibody heavy chain variable region gene PCR system and A7 mouse antibody heavy chain variable region gene PCR system. , A8 mouse antibody heavy chain variable region gene PCR system, A9 mouse antibody heavy chain variable region gene PCR system, A10 mouse antibody heavy chain variable region gene PCR system, A11 mouse antibody heavy chain variable region gene PCR system, A12 mouse antibody heavy chain variable region gene PCR system, A13 mouse antibody heavy chain variable region gene PCR system, A14 mouse antibody heavy chain variable region gene PCR system, A15 mouse antibody heavy chain variable Region gene PCR system, A16 mouse antibody heavy chain variable region gene PCR system, A17 mouse antibody heavy chain variable region gene PCR system, A18 mouse antibody heavy chain variable region gene PCR system, A19 mouse antibody heavy chain Variable region gene 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 small 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 gene PCR system, A34 mouse antibody heavy chain variable region gene PCR system.

The upstream primers of these 29 PCR systems are all HM mix; the cDNA and downstream index primers of these 29 PCR systems are different, among which: the cDNA in the A6 mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A6 , the Barcode sequence contained in the downstream index primer is B6, 5'-CGAAGGTT-3', the cDNA in the A7 mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A7, and the Barcode sequence contained in the downstream index primer is B7 , 5'-CACGCATT-3', A8 The cDNA in the mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A8, and the Barcode sequence contained in the downstream index primer is B8, 5'-TACCAGGT-3', A9 The cDNA in the mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A9. The Barcode sequence contained in the downstream index primer is B9, 5'-TTACCGAG-3', and the A10 mouse antibody heavy chain variable region PCR system The cDNA in is the cDNA of hybridoma cell A10. The Barcode sequence contained in the downstream index primer is B10, 5'-AGTCACCT-3'. The cDNA in the A11 mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A11. , the Barcode sequence contained in the downstream index primer is B11, 5'-TCGCATTC-3', the cDNA in the A12 mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A12, and the Barcode sequence contained in the downstream index primer is B12 , 5'-CATGGTAC-3', A13 The cDNA in the mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A13, and the Barcode sequence contained in the downstream index primer is B13, 5'-CACGGACT-3', A14 The cDNA in the mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A14. The Barcode sequence contained in the downstream index primer is B14, 5'-CTGTAGCA-3', and the A15 mouse antibody heavy chain variable region PCR system The cDNA in is the cDNA of hybridoma cell A15. The Barcode sequence contained in the downstream index primer is B15, 5'-AGAGCTTG-3'. The cDNA in the A16 mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A16. , the Barcode sequence contained in the downstream index primer is B16, 5'-AGCATAGC-3', the cDNA in the A17 mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A17, and the Barcode sequence contained in the downstream index primer is B17 , 5'-CCTACCAT-3', A18 mouse antibody heavy chain variable region cDNA in the PCR system is the cDNA of hybridoma cell A18, and the Barcode sequence contained in the downstream index primer is B18, 5'-GTCGTTGA-3', A19 The cDNA in the mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A19. The Barcode sequence contained in the downstream index primer is B19, 5'-TGCGTATC-3', and the A20 mouse antibody heavy chain variable region PCR system The cDNA in is the cDNA of hybridoma cell A20. The Barcode sequence contained in the downstream index primer is B20, 5'-AGCTAACC-3'. The cDNA in the A21 mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A21. , the Barcode sequence contained in the downstream index primer is B21, 5'-GAACGGTA-3', the cDNA in the A22 mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A22, and the Barcode sequence contained in the downstream index primer is B22 , 5'-GGATTGTC-3', A23 The cDNA in the mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A23, and the Barcode sequence contained in the downstream index primer is B23, 5'-TACCGTCT-3', A24 The cDNA in the mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A24. The Barcode sequence contained in the downstream index primer is B24, 5'-CGAGTATC-3', and the A25 mouse antibody heavy chain variable region PCR system The cDNA in is the cDNA of hybridoma cell A25. The Barcode sequence contained in the downstream index primer is B25, 5'-TGTACGCT-3'. The cDNA in the A16 mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A26. , the Barcode sequence contained in the downstream index primer is B26, 5'-TCCTCTGA-3', the cDNA in the A27 mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A27, and the Barcode sequence contained in the downstream index primer is B27 , 5'-AGCGTATC-3', A28 mouse antibody heavy chain variable region cDNA in the PCR system is the cDNA of hybridoma cell A28, and the Barcode sequence contained in the downstream index primer is B28, 5'-TCATCACC-3', A29 The cDNA in the mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A29. The Barcode sequence contained in the downstream index primer is B29, 5'-GACAGTAC-3', and the A30 mouse antibody heavy chain variable region PCR system The cDNA in is the cDNA of hybridoma cell A30. The Barcode sequence contained in the downstream index primer is B30 and 5'-GTCCTTAC-3'. The cDNA in the A31 mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A31. , the Barcode sequence contained in the downstream index primer is B31, 5'-GGAGCAAT-3', the cDNA in the A32 mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A32, and the Barcode sequence contained in the downstream index primer is B32 , 5'-AGGAACAG-3', A33 The cDNA in the mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A33, and the Barcode sequence contained in the downstream index primer is B33, 5'-CAGTGCTA-3', A34 The cDNA in the mouse antibody heavy chain variable region PCR system is the cDNA of hybridoma cell A34, and the Barcode sequence contained in the downstream index primer is B34, 5'-CTAGCTAG-3'.

Mouse antibody heavy chain variable region gene PCR system: 1 μL of cDNA from one of the A6-A34 hybridoma cells in step 1, 1 μL of mouse antibody heavy chain upstream primer (HM mix), and downstream index primer (containing The specific Barcode sequence corresponds to the cDNA of different hybridoma cells one-to-one) 1 μL, 2×Trans Taq HiFi PCR Super MixⅡ 5 μL, ddH ₂ O 2 μL, the total volume is 10 μL.

B. Antibody light chain variable region gene PCR system:

The only difference between the rat antibody light chain variable region PCR system and the mouse antibody light chain variable region PCR system is that the cDNA template, upstream primer and downstream index primer are different, and the rest are the same.

The rat antibody light chain variable region gene PCR system has a total of 5 PCR systems. These 5 PCR systems are named A1 rat antibody light chain variable region gene PCR system and A2 rat antibody light chain variable region gene PCR system. , A3 rat antibody light chain variable region gene PCR system, A4 rat antibody light chain variable region gene PCR system and A5 rat antibody light chain variable region gene PCR system. The upstream primers of these five PCR systems are all LR mix; the cDNA and downstream index primers of these five PCR systems are all different, among which: the cDNA in the A1 rat antibody light chain variable region PCR system is the cDNA of hybridoma cell A1 , the Barcode sequence contained in the downstream index primer is B35, 5'-TGCAAGCA-3', the cDNA in the A2 rat antibody light chain variable region PCR system is the cDNA of hybridoma cell A2, and the Barcode sequence contained in the downstream index primer is B36 , 5'-CGCATCTA-3', A3 The cDNA in the rat antibody light chain variable region PCR system is the cDNA of hybridoma cell A3, and the Barcode sequence contained in the downstream index primer is B37, 5'-CGTATCTG-3', A4 The cDNA in the rat antibody light chain variable region PCR system is the cDNA of hybridoma cell A4. The Barcode sequence contained in the downstream index primer is B38, 5'-GTAGACCT-3', and the A5 rat antibody light chain variable region PCR system The cDNA in is the cDNA of hybridoma cell A5, and the Barcode sequence contained in the downstream index primer is B39, 5'-CGCCATTA-3'.

Rat antibody light chain variable region gene PCR system: 1 μL of cDNA from one of the A1-A5 hybridoma cells in step 1, 1 μL of rat antibody light chain upstream primer (LR mix), and downstream index primer (containing The specific Barcode sequence corresponds to the cDNA of different hybridoma cells one-to-one) 1 μL, 2×Trans Taq HiFi PCR Super Mix II 5 μL, ddH ₂ O 2 μL, the total volume is 10 μL.

The mouse antibody light chain variable region gene PCR system has a total of 29 PCR systems. These 5 PCR systems are named A6 mouse antibody light chain variable region gene PCR system and A7 mouse antibody light chain variable region gene PCR system. , A8 mouse antibody light chain variable region gene PCR system, A9 mouse antibody light chain variable region gene PCR system, A10 mouse antibody light chain variable region gene PCR system, A11 mouse antibody light chain variable region gene PCR system, A12 mouse antibody light chain variable region gene PCR system, A13 mouse antibody light chain variable region gene PCR system, A14 mouse antibody light chain variable region gene PCR system, A15 mouse antibody light chain variable Region gene PCR system, A16 mouse antibody light chain variable region gene PCR system, A17 mouse antibody light chain variable region gene PCR system, A18 mouse antibody light chain variable region gene PCR system, A19 mouse antibody light chain Variable region gene 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 small 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 gene PCR system, A34 mouse antibody light chain variable region gene PCR system system.

The upstream primers of these 29 PCR systems are all LM mix; the cDNA and downstream index primers of these 29 PCR systems are different, among which: the cDNA in the A6 mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A6 , the Barcode sequence contained in the downstream index primer is B40, 5'-ATGACACC-3', the cDNA in the A7 mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A7, and the Barcode sequence contained in the downstream index primer is B41 , 5'-CAAGTTGA-3', A8 The cDNA in the mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A8, and the Barcode sequence contained in the downstream index primer is B42, 5'-TGCTGAAG-3', A9 The cDNA in the mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A9. The Barcode sequence contained in the downstream index primer is B43, 5'-ATGACCGT-3', and the A10 mouse antibody light chain variable region PCR system The cDNA in is the cDNA of hybridoma cell A10. The Barcode sequence contained in the downstream index primer is B44, 5'-TGGCGAGA-3'. The cDNA in the A11 mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A11. , the Barcode sequence contained in the downstream index primer is B45, 5'-GCACTTCC-3', the cDNA in the A12 mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A12, and the Barcode sequence contained in the downstream index primer is B46 , 5'-GTTACCGA-3', A13 mouse antibody light chain variable region cDNA in the PCR system is the cDNA of hybridoma cell A13, and the Barcode sequence contained in the downstream index primer is B47, 5'-GACGTCAC-3', A14 The cDNA in the mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A14. The Barcode sequence contained in the downstream index primer is B48, 5'-GTTACAGC-3', and the A15 mouse antibody light chain variable region PCR system The cDNA in is the cDNA of hybridoma cell A15. The Barcode sequence contained in the downstream index primer is B49, 5'-TCGATTGC-3'. The cDNA in the A16 mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A16. , the Barcode sequence contained in the downstream index primer is B50, 5'-GACGATCT-3', the cDNA in the A17 mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A17, and the Barcode sequence contained in the downstream index primer is B51 , 5'-TGACGCTA-3', A18 The cDNA in the mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A18, and the Barcode sequence contained in the downstream index primer is B52, 5'-ATAACGCG-3', A19 The cDNA in the mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A19. The Barcode sequence contained in the downstream index primer is B53, 5'-GATTCACC-3', and the A20 mouse antibody light chain variable region PCR system The cDNA in is the cDNA of hybridoma cell A20. The Barcode sequence contained in the downstream index primer is B54, 5'-GGCAAGTA-3'. The cDNA in the A21 mouse antibody light chain variable region PCR system is the cDNA of 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 hybridoma cell A22, and the Barcode sequence contained in the downstream index primer is B56 , 5'-CATTGCGA-3', A23 The cDNA in the mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A23, and the Barcode sequence contained in the downstream index primer is B57, 5'-GCTGTACA-3', A24 The cDNA in the mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A24. The Barcode sequence contained in the downstream index primer is B58, 5'-TAGCAGCT-3', and the A25 mouse antibody light chain variable region PCR system The cDNA in is the cDNA of hybridoma cell A25. The Barcode sequence contained in the downstream index primer is B59 and 5'-AGCGACAT-3'. The cDNA in the A26 mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A26. , the Barcode sequence contained in the downstream index primer is B60, 5'-CATTAGCC-3', the cDNA in the A27 mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A27, and the Barcode sequence contained in the downstream index primer is B61 , 5'-CATCTCCA-3', A28 mouse antibody light chain variable region cDNA in the PCR system is the cDNA of hybridoma cell A28, and the Barcode sequence contained in the downstream index primer is B62, 5'-GTCATCGT-3', A29 The cDNA in the mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A29. The Barcode sequence contained in the downstream index primer is B63, 5'-GGACTTCA-3', and the A30 mouse antibody light chain variable region PCR system The cDNA in is the cDNA of hybridoma cell A30. The Barcode sequence contained in the downstream index primer is B64 and 5'-CAGGATCA-3'. The cDNA in the A31 mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A31. , the Barcode sequence contained in the downstream index primer is B65, 5'-GAACGATG-3', the cDNA in the A32 mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A32, and the Barcode sequence contained in the downstream index primer is B66 , 5'-CGACTTGC-3', A33 The cDNA in the mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A33, and the Barcode sequence contained in the downstream index primer is B67, 5'-GATTCCGT-3', A34 The cDNA in the mouse antibody light chain variable region PCR system is the cDNA of hybridoma cell A34, and the Barcode sequence contained in the downstream index primer is B68 and 5'-CCGCTTAC-3'.

Mouse antibody light chain variable region gene PCR system: 1 μL of cDNA from one hybridoma cell in the A6-A34 hybridoma cells in step 1, 1 μL of mouse antibody light chain upstream primer (LM mix), and downstream index primer (containing The specific Barcode sequence corresponds to the cDNA of different hybridoma cells one-to-one) 1 μL, 2×Trans Taq HiFi PCR Super MixⅡ 5 μL, ddH ₂ O 2 μL, the total volume is 10 μL.

C. The reaction conditions of the PCR system are as follows: pre-denaturation at 94°C for 5 minutes; then 30 cycles as follows: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds; extension at 72°C for 7 minutes, and storage at 4°C.

3) Amplification product detection: Take 5 μL of the PCR product obtained in step 2) of this sequencing method, and use 1.5% agarose gel electrophoresis to detect the PCR product.

4) Sequencing analysis: Mix the heavy chain variable region gene PCR products and light chain variable region gene PCR products of all antibodies to be tested, and use a gel recovery kit to recover the products. The recovered products were sent to Novogene for high-throughput sequencing.

5) Data analysis: The sequencing result data obtained in step 4) is analyzed using the antibody sequence analysis program developed by the company. The flow chart of the antibody sequence analysis program is shown in Figure 2. The data analysis program design ideas are as follows:

A. Basic structure of antibody sequence

(1) The antibody heavy chain from N-terminus to C-terminus is randomly generated by one of the VH sequences in several genomes, one of the DH sequences in several genomes, and one of the JH sequences in several genomes. The variable region formed by connecting the Junction1H sequence and Junction2H sequence forms the following structure VH-Junction1H-DH-Junction2H-JH;

(2) The antibody light chain from N-terminus to C-terminus is composed of one of the VL sequences in several genomes, one of the JL sequences in several genomes, and a variable region connected by a randomly generated JunctionL sequence in the middle. The following structure VL-JunctionL-JL.

B. Heavy chain sequence analysis ideas

(1) Establish heavy chain databases VH, DH, JH;

(2) Compare the C-terminal sequence with the JH database to determine the JH fragment;

(3) Compare the N-terminal sequence with the VH database to determine the VH fragment;

(4) Remove the JH fragment and VH fragment, compare the remaining sequence with the database DH, and determine the DH fragment and the randomly generated Junction1H sequences and Junction2H at both ends.

C. Ideas for light chain sequence analysis

(1) Establish light chain database VL, JL;

(2) Compare the C-terminal sequence with the JL database to determine the JL fragment;

(3) Compare the N-terminal sequence with the VL database to determine the VL fragment;

(4) Remove the JL fragment and VL fragment to obtain a randomly generated JunctionL sequence.

D. Tracking the evolution of heavy and light chains

Because antibody heavy and light chains continue to mutate during immunity, antibody evolution can be tracked. Specific steps are as follows:

(1) Extract heavy chain sequences with the same basic structure, that is, sequences with the same VH fragment, DH fragment, and JH fragment, and use the mutation-free genome sequence as the origin to establish a heavy chain evolutionary tree through the ClustalOmega program;

(2) Extract light chain sequences with the same basic structure, that is, sequences with the same VL fragment and JL fragment, and use the mutation-free genome sequence as the origin to build a light chain evolutionary tree through the ClustalOmega program.

Barcode-containing primers were used to amplify 5 rat antibodies and 29 mouse antibodies. The results are shown in Figure 3 and Figure 4: Use barcode-containing primers to amplify the antibody light chain variable region gene and heavy chain variable region gene, PCR The products all amplified bands between 250bp and 500bp, and the band size was consistent with the predicted size.

Mix the barcode-containing PCR products and perform next-generation sequencing. The sequencing results were analyzed using software to obtain the correct light chain and heavy chain variable region gene sequences of 5 rat antibodies and 29 mouse antibodies.

The antibody sequencing results are as follows: all antibodies were sequenced successfully. For the nucleotide sequence of the detailed sequencing results, see Sequence 66 to Sequence 133 in the sequence listing. Among them: the light chain variable region gene sequence of antibody A1 is the DNA molecule of sequence 66 in the sequence listing, and the heavy chain variable region gene sequence is the DNA molecule of sequence 67 in the sequence listing; the light chain variable region gene sequence of antibody A2 is The DNA molecule of sequence 68 in the sequence listing, the heavy chain variable region gene sequence is the DNA molecule of sequence 69 in the sequence listing; the light chain variable region gene sequence of antibody A3 is the DNA molecule of sequence 70 in the sequence listing, and the heavy chain variable region The region gene sequence is the DNA molecule of sequence 71 in the sequence listing; the light chain variable region gene sequence of antibody A4 is the DNA molecule of sequence 72 in the sequence listing, and the heavy chain variable region gene sequence is the DNA molecule of sequence 73 in the sequence listing; The light chain variable region gene sequence of antibody A5 is the DNA molecule of sequence 74 in the sequence list, and the heavy chain variable region gene sequence is the DNA molecule of sequence 75 in the sequence list; the light chain variable region gene sequence of antibody A6 is the sequence list. The DNA molecule of sequence 76, the heavy chain variable region gene sequence is the DNA molecule of sequence 77 in the sequence list; the light chain variable region gene sequence of antibody A7 is the DNA molecule of sequence 78 in the sequence list, and the heavy chain variable region gene The sequence is the DNA molecule of sequence 79 in the sequence listing; the light chain variable region gene sequence of antibody A8 is the DNA molecule of sequence 80 in the sequence listing, and the heavy chain variable region gene sequence is the DNA molecule of sequence 81 in the sequence listing; antibody A9 The light chain variable region gene sequence is the DNA molecule of sequence 82 in the sequence listing, and the heavy chain variable region gene sequence is the DNA molecule of sequence 83 in the sequence listing; the light chain variable region gene sequence of antibody A10 is the sequence in the sequence listing The DNA molecule of 84, the heavy chain variable region gene sequence is the DNA molecule of sequence 85 in the sequence listing; the light chain variable region gene sequence of antibody A11 is the DNA molecule of sequence 86 in the sequence listing, and the heavy chain variable region gene sequence is The DNA molecule of sequence 87 in the sequence listing; the light chain variable region gene sequence of antibody A12 is the DNA molecule of sequence 88 in the sequence listing, and the heavy chain variable region gene sequence is the DNA molecule of sequence 89 in the sequence listing; the light chain variable region gene sequence of antibody A13 The gene sequence of the chain variable region is the DNA molecule of sequence 90 in the sequence listing, and the gene sequence of the heavy chain variable region is the DNA molecule of sequence 91 in the sequence listing; the light chain variable region gene sequence of antibody A14 is the DNA molecule of sequence 92 in the sequence listing. DNA molecule, the heavy chain variable region gene sequence is the DNA molecule with sequence 93 in the sequence listing; the light chain variable region gene sequence of antibody A15 is the DNA molecule with sequence 94 in the sequence listing, and the heavy chain variable region gene sequence is the sequence listing The DNA molecule of sequence 95 in the sequence list; the light chain variable region gene sequence of antibody A16 is the DNA molecule of sequence 96 in the sequence listing, and the heavy chain variable region gene sequence is the DNA molecule of sequence 97 in the sequence listing; the light chain of antibody A17 can be The variable region gene sequence is the DNA molecule with sequence 98 in the sequence list, the heavy chain variable region gene sequence is the DNA molecule with sequence 99 in the sequence list; the light chain variable region gene sequence of antibody A18 is the DNA molecule with sequence 100 in the sequence list , the heavy chain variable region gene sequence is the DNA molecule of sequence 101 in the sequence listing; the light chain variable region gene sequence of antibody A19 is the DNA molecule of sequence 102 in the sequence listing, and the heavy chain variable region gene sequence is the sequence in the sequence listing The DNA molecule of 103; the light chain variable region gene sequence of antibody A20 is the DNA molecule of sequence 104 in the sequence listing; the heavy chain variable region gene sequence is the DNA molecule of sequence 105 in the sequence listing; the light chain variable region of antibody A21 The gene sequence is the DNA molecule of sequence 106 in the sequence listing, and the heavy chain variable region gene sequence is the DNA molecule of sequence 107 in the sequence listing; the light chain variable region gene sequence of antibody A22 is the DNA molecule of sequence 108 in the sequence listing, and the heavy chain variable region gene sequence is the DNA molecule of sequence 107 in the sequence listing. The gene sequence of the chain variable region is the DNA molecule of sequence 109 in the sequence list; the gene sequence of the light chain variable region of antibody A23 is the DNA molecule of sequence 110 in the sequence list, and the gene sequence of the heavy chain variable region is the DNA molecule of sequence 111 in the sequence list. DNA molecule; the light chain variable region gene sequence of antibody A24 is the DNA molecule with sequence 112 in the sequence listing, and the heavy chain variable region gene sequence is the DNA molecule with sequence 113 in the sequence listing; the light chain variable region gene sequence of antibody A25 is the DNA molecule with sequence 114 in the sequence listing, and the heavy chain variable region gene sequence is the DNA molecule with sequence 115 in the sequence listing; the light chain variable region gene sequence of antibody A26 is the DNA molecule with sequence 116 in the sequence listing, and the heavy chain can be The variable region gene sequence is the DNA molecule of sequence 117 in the sequence list; the light chain variable region gene sequence of antibody A27 is the DNA molecule of sequence 118 in the sequence list, and the heavy chain variable region gene sequence is the DNA molecule of sequence 119 in the sequence list. ; The light chain variable region gene sequence of antibody 28 is the DNA molecule of sequence 120 in the sequence listing, and the heavy chain variable region gene sequence is the DNA molecule of sequence 121 in the sequence listing; the light chain variable region gene sequence of antibody A29 is sequence The DNA molecule of sequence 122 in the list, the heavy chain variable region gene sequence is the DNA molecule of sequence 123 in the sequence listing; the light chain variable region gene sequence of antibody A30 is the DNA molecule of sequence 124 in the sequence listing, and the heavy chain variable region The gene sequence is the DNA molecule of sequence 125 in the sequence listing; the light chain variable region gene sequence of antibody A31 is the DNA molecule of sequence 126 in the sequence listing, and the heavy chain variable region gene sequence is the DNA molecule of sequence 127 in the sequence listing; the antibody The light chain variable region gene sequence of A32 is the DNA molecule with sequence 128 in the sequence listing, and the heavy chain variable region gene sequence is the DNA molecule with sequence 129 in the sequence listing; the light chain variable region gene sequence of antibody A33 is the DNA molecule in sequence listing The DNA molecule of sequence 130, the heavy chain variable region gene sequence is the DNA molecule of sequence 131 in the sequence listing; the light chain variable region gene sequence of antibody A34 is the DNA molecule of sequence 132 in the sequence listing, and the heavy chain variable region gene sequence It is the DNA molecule of sequence 133 in the sequence listing.

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art.

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.