Soluble Expression And Detection Of Single-chain Antibodies Against H9N2 Subtype Of Avian Influenza Virus In E.Coli

In recent years,the growing bird flu epidemic caused by the Avian Influenza Virus(AIV)has caused huge economic losses to the poultry industry in China and other countries and has seriously threatened human health.H9N2 is a subtype of AIV that is not only widespread among poultry worldwide,but also can infect humans.It is also a donor of H5N1 AIV internal genes in the 1997 bird flu incident in Hong Kong.In the study of H9N2 prevention and immunity,the single chain antibody fragment(scFv)against the virus is favored because of its small molecular weight,strong penetrability,and strong binding to antigens.In this study,the scFv sequences of PB2 protein targeting H9N2 AIV were obtained from the NCBI gene bank,and the single-chain antibody of H9N2 AIV was prepared using E.coli with natural expression advantages.Due to the unique structure of scFv itself,its expression product in E.coli is essentially in the form of inclusion bodies.After the process of denaturation and renaturation,the protein activity is extremely low.In this experiment,H9N2 AIV scFv was promoted to express soluble in E.coli through genetic engineering methods combined with optimization of expression conditions and process optimization,and its binding ability to antigen PB2 was initially detected.In this experiment,primers for the H9N2 AIV scFv gene sequence were first synthesized.The H9N2 AIV scFv gene fragment sequence was obtained by indirect PCR and constructed into the pET23a-T and pET23a-sfGFP-MAN vectors to obtain the recombinant plasmid pET23a-scFv,pET23a-sfGFP-scFv.The recombinant plasmid was transformed into E.coli Rosetta Blue strain and induced to express.After the H9N2 AIV scFv gene was fused with super fold green fluorescent protein(sfGFP)by SDS-PAGE,the scFv was partially soluble.But the ruslt is negative when not fused with sfGFP,even if the scFv was changed in temperature,inducer concentration,and culture period,no soluble expression was observed.In this experiment,the PB2 gene of H9N2AIV was synthesized by nested PCR and cloned into the eukaryotic expression vector pcDNA-DsRed to obtain the recombinant eukaryotic expression plasmid pcDNA-PB2-DsRed.The recombinant plasmid was transfected by Lipfectin3000.The PB2 protein was expressed in HEK293T cells.The scFv expressed in E.coli was extracted and purified,Western Blot and cellular immunofluorescence experiments were performed on HEK293T cells transfected with pcDNA-PB2-DsRed.The results revealed that the PB2 protein can be specifically recognized by soluble scFv obtained from E.coli.It was shown that soluble scFv obtained from E.coli have biological activity against H9N2 AIV,which laid a theoretical foundation for further establishing emergency immunization and detection diagnostic methods with this single chain antibody.