Construction, Screening And Gene Expression Of A Phage-Displayed Scfv Antibody Library Against Swine Flu And

Swine influenza (swine influenza, SI) is a common respiratory disease of swine throughout the world caused by swine influenza virus (SIV). Once occurs, it may cause economic lost to swine industry, furthermore, co-infecting with other respiratory diseases. Pigs are susceptible to avian, human influenza viruses and have been proposed to be intermediate hosts or"mixing vessels" for the generation of pandemic influenza viruses. Swine influenza dose not only have severe veterinary importance, but also poses serious public health threats. Rencently, immunity is the main measure of prevention of SI, but SI is still epidemic because of many reasons, such as the interference of maternal antibody, no cross-reaction between many virus subtypes and so on, so the conventional vaccination mesures are sometimes powerless. Therefore, we can explore new measures of diseases prevention from disease-resistant breeding. In this study, we sreen scFv from phage antibody libraries against H1N1subtype swine influenza virus. We could get genotype strain of the antibody, then we do research about transgenic pig against swine influenza virus.Test Ⅰ Building and optimization the culture of H1N1subtype swine influenza virus in vitro. H1N1subtype swine influenza virus was cultured in MDCK cells. Firstly we optimized the culture of MDCK cells by comparing the growth of cells under different serum concentrations. MDCK cells infected by swine influenza virus were seen by immunofluoresence. We found the best serum concentration, the best TPCK trypsin concentration and the best time of incubation through hemagglutination test. The result indicated that the best serum concentration of cells culture is10%, H1N1subtype swine influenza virus was cultured72h well in the serum-free medium containing1.5p,g/mL TPCK trypsin.Test Ⅱ Construction and screening of recombinant monoclonal antibody anti-swine influenza using phage display technology. The BALB/C mice were immuned by swine flu virus, and the total RNA were extracted from spleen. The first strand of cDNA was amplified by RT-PCR, and was taken as template. The heavy chain and light chain variable region genes (VH and VL) were amplified respectively by PCR. They were linked by a DNA linker encoding (Gly4Ser)3as VH-linker-VL sequence forming scFv by SOE(splicing by overlap extension) PCR. The scFv gene was enzymed by sfiⅠ and Not Ⅰ. It was connected to the vector of pCANTAB5E. The recombinant vector was transformed into the susceptible Escherichia coli TGI, then was rescued with the help of phage M13K07, a library of phage scFv antibody was constructed. The swine flu virus was coated on96well plate, after three rounds of enrichment screening and the identification of Phage-ELISA, the scFv antibody library against swine flu was constructed and the capacity of the library was4×106cfu/mL. Four strain of scFv antibodies anti-the swine flu virus were screened from the library. They could combine swine flu incompetition with positive polyclonal antibody from mouse, one of them named as WJY001had higher binding reaction with H1N1subtype of swine influenza virus.Test Ⅲ The indication of single-chain antidoies of WJY001. First, the size and sequence of scFv was indentified by PCR and sequence analysis. Then, we did blocking ELISA and sandwich ELISA, so we were sure whether the scFv of WJY001could combine with H1N1subtype of swine influenza virus, competing with positive antibody from mice. Finally, we did experiments on cells:virus blocking experiments, and the scFv was transfected into A549cells, then we observed whether the scFv expressed in the A549cells by immunofluorescence. The result indicated that the scFv of WJY001could combine with H1N1subtype of swine influenza virus, competing with positive antibody from mice, and the infection of A549cells by swine influenza virus could be stopped by the scFv of WJY001. The scFv of WJY001could be expressed in the A549cells and could block the infection of H1N1subtype of swine influenza virus.Test Ⅳ Expression of single-chain antidoies of WJY001. The scFv of WJY001was amplified by PCR, it was connected to the vector of PET28a, The recombinant vector was transformed into the susceptible Escherichia coli BL21, and was induced by IPTG, finally the products was indicated by Western Blot and ELISA. The results indicated that single-chain antibodies mainly expressed in precipitation. Single-chain antibodies could combine swine influenza virus after refolding.