Preparation method for single-chain antibody based on hybridoma cell

A hybridoma cell and single-chain antibody technology, which is applied in the field of single-chain antibody preparation, can solve the problems of increased blindness, difficult display efficiency, complex and inefficient preparation process of single-chain antibody, etc.

Active Publication Date: 2013-06-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to screen the antibody gene process in the prior art according to defects such as blindness, pseudo-antibody genes...

Method used

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  • Preparation method for single-chain antibody based on hybridoma cell
  • Preparation method for single-chain antibody based on hybridoma cell
  • Preparation method for single-chain antibody based on hybridoma cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Preparation of single-chain antibody against RAC

[0046] 1. Cloning of Anti-RAC Antibody Variable Region Genes

[0047] RAC monoclonal hybridoma cell AC2 (preserved in our laboratory) was revived, and cultured in RPMI 1640 complete medium to 1×10 7 , Total RNA was extracted with Trizol (TAKARA Company) according to the manufacturer's instructions.

[0048] The total RNA was reverse-transcribed into the first strand of cDNA using a reverse transcription kit (Promega), following the manufacturer's instructions.

[0049] Use general-purpose primers to clone antibody heavy and light chain variable region genes. The general-type primers are shown in Table 1. Taking the light chain variable region gene cloning as an example, first mix the 6 primers of LF in equal proportions, and mix the LF primers separately. Combined with 19 primers from LB1~19 to form 19 primer sets, using the first strand of cDNA as a template, these 19 primer sets were used to amplify the ...

Embodiment 2

[0075] Example 2 Anti-furaltadone metabolites (Furazolidone Metabolite , AMOZ) single-chain antibody preparation

[0076] 1. Cloning of Anti-AMOZ Antibody Variable Region Gene

[0077] AMOZ monoclonal hybridoma cell 2BE (preserved in our laboratory) was revived and cultured in RPMI 1640 complete medium to 1×10 7 , Total RNA was extracted with Trizol (TAKARA Company) according to the manufacturer's instructions.

[0078] The total RNA was reverse-transcribed into the first strand of cDNA using a reverse transcription kit (Promega), following the manufacturer's instructions.

[0079] The heavy and light chain variable region genes of the AMOZ antibody were cloned using general-type degenerate primers. The general-type degenerate primers are shown in Table 1. The primer set screening method and the antibody gene cloning method were the same as in Example 1.

[0080] The screened primer combinations are: HB1-HF3, HB9-HF3, HB15-HF3, HB16-HF6, HB17-HF6, LB18-LF5, LB10-LF6, LB1...

Embodiment 3

[0106] Example 3 Preparation of single-chain antibody against OPs

[0107] 1. Cloning of Antibody Variable Region Genes Against OPs

[0108] After resuscitating OPs monoclonal hybridoma cell 12C2 (preserved in our laboratory), expand the culture in RPMI 1640 complete medium to 1×10 7 , Total RNA was extracted with Trizol (TAKARA Company) according to the manufacturer's instructions.

[0109] The total RNA was reverse-transcribed into the first strand of cDNA using a reverse transcription kit (Promega), following the manufacturer's instructions.

[0110] The heavy and light chain variable region genes of the OPs antibody were cloned using general-type degenerate primers. The general-type degenerate primers are shown in Table 1. The primer set screening method and the antibody gene cloning method were the same as in Example 1.

[0111] The screened primer combinations are: HB1-HF4, HB8-HF4, HB9-HF4, HB12-HF4, HB15-HF4, HB18-HF4, LB1-LF1, LB5-LF2, LB11-LF1, LB17-LF1, LB17-LF2...

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Abstract

The invention discloses a preparation method for a single-chain antibody based on a hybridoma cell. The preparation method comprises the following steps: carrying out inverse transcription with total mRNA of the hybridoma cell as a template to obtain cDNA and selecting and using universal primers with low degeneracy for amplification of a complete set of heavy chain variable region and light chain variable region genes of the antibody; inputting sequences of the amplified heavy chain and light chain variable region genes into an IMGT database for analysis and eliminating antibody genes and nonsense genes originated from the hybridoma cell so as to obtain candidate heavy chain and light chain variable region genes; translating the genes into amino acid sequences, carrying out three dimensional structural homology modeling to obtain a three dimensional simulation structure of the single-chain antibody and carrying out molecular docking analysis on the three dimensional simulation structure and a corresponding antigen target molecule; and splicing a candidate heavy chain and a candidate light chain with correct analysis results into a single-chain antibody gene by using an overlap extension PCR method and carrying out expression and identification of antibody activity. The preparation method provided by the invention is applicable to preparation of all the single-chain antibodies originated from monoclonal hybridoma cells; the process of the preparation method is simple and rapid, and a prepared product has high quality.

Description

technical field [0001] The invention relates to the fields of immunology and molecular biology, in particular to a method for preparing a hybridoma cell-based single-chain antibody. Background technique [0002] In the early 1980s, with the development of molecular biology, people began to use genetic engineering technology to produce recombinant antibodies, which opened up a new era of antibody application in a wide range of fields, among which the research on single-chain antibody (single-chain antibody, scFv) made attention. Single-chain antibodies are composed of a linker peptide (linker) to the antibody heavy chain variable region V H and light chain variable region V L The single peptide chain formed by joining together, through correct folding, the two variable regions can form an Fv segment with antigen binding function through non-covalent bonds. Due to its advantages such as short preparation period, various tags can be added for purification, and easy genetic...

Claims

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Application Information

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IPC IPC(8): C12N15/13C12N15/63C12N5/20C07K16/00C07K16/44
Inventor 孙远明董洁娴王弘雷红涛李振峰徐振林杨金易沈玉栋
Owner SOUTH CHINA AGRI UNIV
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