Method for obtaining human antibody variable region by combination of SOLID sequencing and single-cell RT-PCR

Abstract

The present invention provides a method for obtaining a human antibody variable region by combination of SOLID sequencing and single-cell RT-PCR. According to the method, a cell surface molecular marker is adopted to screen a single cell expressing a specific antibody; mRNA of the single cell is subjected to a reverse transcription to obtain a cDNA sequence; then a designed specific IgG / M primer group is adopted to carry out a PCR amplification; the resulting amplification product is adopted as a template, and SOLID sequencing primers (Barcoded Primers) are adopted to carry out one PCR amplification; and finally the resulting amplification product by adopting the Barcoded Primers is adopted as a template, SOLID sequencing is performed, and the sequencing result is subjected to bioinformatics analysis to obtain a human antibody variable region gene sequence. The method of the present invention can be widely used for obtaining variable region sequences of heavy chains and light chains of specific antibodies in a variety of diseases, and advantages of rapidness and efficiency of the method provide an effective means for production of fully humanized antibody drugs.

Description

technical field [0001] The invention relates to the technical field of obtaining human specific antibody variable region sequences, in particular to a method for obtaining human antibody variable region sequences combined with SOLID sequencing and single-cell RT-PCR. Background technique [0002] From the large-scale outbreak of SARS in 2002, to the hand, foot and mouth disease caused by the EV71 virus in 2008, to the global influenza pandemic caused by the H1N1 influenza virus in Mexico in 2009, once they broke out, they quickly spread on a large scale or globally . The development of conventional antiviral drugs is a long process, which cannot meet the needs in a short time. Moreover, the rapid mutation of these infectious bacteria makes it increasingly difficult for us to find effective prevention and control methods in a short period of time. For the explosive and epidemic diseases caused by such pathogens, it is very urgent to find effective prevention and treatment m...

AI Technical Summary

Problems solved by technology

[0007] In the above-mentioned fully humanized antibody technology, the preparation and production process is complicated, and it takes a long time and cycle, which is not suitable for the development and production of antibody drugs for some sudden and epidemic diseases, and it is not suitable for the rapid mutation of some pathogens or antigens. appear powerless

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