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Method for constructing natural humanized IgG Fab phage antibody library

A technology of phage antibody library and construction method, which is applied in the field of construction of natural human IgG Ab phage antibody library, and can solve the problem of low actual efficiency of antibody library

Inactive Publication Date: 2010-11-24
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the actual efficiency of the antibody library constructed by this strategy is not high

Method used

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  • Method for constructing natural humanized IgG Fab phage antibody library
  • Method for constructing natural humanized IgG Fab phage antibody library
  • Method for constructing natural humanized IgG Fab phage antibody library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Separation of human peripheral blood lymphocytes and extraction of total RNA

[0058] 2 to 300 samples of anticoagulated peripheral blood were collected in batches from 2 to 300 healthy volunteers aged 20 to 40 years old who had not suffered from infectious diseases such as colds for nearly 1 to 6 months, and were separated with lymphocyte separation medium to obtain peripheral blood. The blood lymphocytes were mixed, washed with sterilized PBS and resuspended to obtain about 8×108 cells in total. The total RNA of peripheral blood lymphocytes was extracted with the total RNA purification kit.

Embodiment 2

[0059] Example 2 Amplification of human immunoglobulin Fd heavy chain and light chain genes

[0060] RT-PCR with Gene Amp RNA PCR Kit: First, reverse-transcribe the total lymphocyte RNA into cDNA, and then use a set of specificity covering almost all light chain or recombination variable region coding sequences of human immunoglobulin G (IgG) Primers were used to perform PCR on the above reaction products to amplify Fd fragments of immunoglobulin kappa, lambda light chain and gamma heavy chain respectively. The amplification conditions were: 94°C for 2 minutes, then 94°C for 1-2 minutes, 50-60°C for 1-2 minutes, 72°C for 1-2 minutes, a total of 30 cycles, and finally extended to 72°C for 1-10 minutes. The PCR products were identified by 2.0% agarose gel electrophoresis, which showed that the length of the heavy chain Fd gene was about 690 bp, and the length of the light chain k and l genes was about 660 bp ( figure 1 ).

Embodiment 3

[0061] Example 3 Construction of Natural Human IgG Fab Phage Antibody Library

[0062] The light chain genes κ and λ were digested with restriction endonucleases Asc I and Nhe I respectively, and after purification, the κ:λ light chain genes were mixed at 7:1 and mixed with the phage vector pFabICN [1] connect( figure 2 ), the ligation product was purified and dissolved in 4 μl distilled water, electroporated twice to JM109 competent cells, and the transformed bacteria were evenly spread on LB plates containing 50 μl ampicillin, and the plasmid DNA of all clones was collected, which was the light chain antibody library. After identification by HindIII single enzyme digestion, the DNA gene size of the light chain antibody library is 5060bp ( image 3 ).

[0063] The PCR product of the Fd fragment gene of the γ heavy chain was digested with Sfi I and Not I, purified and ligated with the DNA of the light chain library, and electrotransformed in the same way to obtain the pla...

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Abstract

The invention relates to the technical field of genetic engineering, in particular a method for constructing a natural humanized IgG Fab phage antibody library. In the method, Fd genes of the whole set of humanized antibody light chains and antibody heavy chains are amplified from peripheral blood lymphocyte of healthy volunteers by DNA recombinant technology, and are inserted into corresponding positions of phage vector pFabICN so as to construct the natural humanized IgG Fab phage antibody library with high capacity and high variety. Experiments prove that the capacity of the phage antibody library constructed by the method can reach as high as 4*108; and the phage antibody library accords with high-capacity antibody library standard, includes Fd genes of the whole set of humanized antibody light chains and heavy chains, and has high variety. Compared with other phage antibody library constructing methods, the antibody library constructed by the method has the advantages of high capacity and high variety, can be used in low-antigenicity high-compatibility humanized antibody for high-flux screening clinical treatment.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing a natural human IgG Fab phage antibody library. Background technique [0002] Genetic engineering antibody library technology is a new method of in vitro preparation of human monoclonal antibody developed in recent years. By constructing and screening phage antibody library, it provides a novel and effective way for the production of scientific research and therapeutic antibodies. The phage antibody library is a collection of diverse phage antibodies obtained by assembling the diverse variable region genes of antibody molecules into expression vectors and expressing them on the surface of phages. Through the enrichment process of "adsorption-elution-amplification", the variable region genes of specific antibodies can be screened out from the phage antibody library very effectively. At present, phage antibody libraries are mainly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C40B40/02C12N15/13C12N15/63
Inventor 程训佳付永峰橘裕司
Owner FUDAN UNIV
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