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Sorting of EIAV envelop protein monoclonal antibodies based on single B cell amplification technology

ZHANG Jia-qi;WANG Ya-yu;GUO Xing;LIN Yue-zhi;LIAO Hua-xin;WANG Xiao-jun;State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences;College of Life Science and Technology, Jinan University;  
Equine infectious anemia virus (EIAV) envelope protein (Env) is a key protein involved in the infection process which consists of surface protein (Gp90) and transmembrane protein (Gp45).Env plays an important part in virus adhesion,cell fusion,disease transmission and other aspects.In order to obtain Env-specific monoclonal antibody (MAb),we constructed the codon-optimized Gp90 gene on pcDNA3.1,and built the recombinant plasmid pcDNA3.1-s-gp90-His.According to the conventional method,after immunizing mice with the recombinant plasmid pcDNA3.1-s-gp90-His 6 times,spleen cells were isolated.We used fluorescence activated cell sorting (FACS) technology to isolate the single B lymphocyte that can recognize Gp90 in the spleen cells of immunized mice.890 single B lymphocytes that recognize Gp90 were obtained from 2×10~8mouse spleen cells.Then,we amplified the variable region genes of the antibody heavy chain (Ig VH) and light chain (Ig VL) through specific primers using the genomic cDNA of single B lymphocyte as a template,and the heavy chain (Ig VH) and the light chain (Ig VL) variable region gene was ligated to pcDNA3.1 by overlapping PCR.The sequencing and identification results showed that 27 pairs of matching pcDNA3.1-VH and pcDNA3.1-VL expression plasmids were successfully constructed.The plasmids were co-transfected into HEK293F cells at the ratio of pcDNA3.1-VH:pcDNA3.1-VL=1:1,and the cell supernatant was harvested and purified by Protein A column to obtain Gp90 monoclonal antibody.The purified Gp90 monoclonal antibodies were identified by ELISA and western blot.Results showed that 4 monoclonal antibodies can bind with Gp90 protein and recognize the natural conformation of Gp90 protein from 27 matched antibody gene pairs were obtained.This study used single B lymphocyte amplification technology to screen Gp90 monoclonal antibodies for the first time.At the same time,the acquisition of Gp90 monoclonal antibodies provided a basis for the development of Env protein detection methods and laid a foundation for in-depth study of the function of Env protein.
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