Prokaryotic Expression Of Feline Infectious Peritonitis Virus N Protein And Preparation Of Rabbit-derived Single-chain Antibody

Feline infectious peritonitis virus（FIPV）is an infectious disease with high virulence and high lethality that causes feline infectious peritonitis,and belongs to Feline Coronavirus（FCo V）.With the increasing number of cat owners,more and more attentions has been paid to the infectious diseases of cats.Furthermore,testing and treatment of Coronavirus are of particular importance,especially for FIPV in the view of its lethality and no functional treatments so far.Single chain fragment variable（sc Fv）has been widely used in immunological diagnosis,antiviral drug target screening and other fields in recent years based on its own advantages.Screening of sc Fv against FIPV can lay the foundation for immunological detection,diagnosis and treatment of feline coronavirus.Therefore,in this study,p ET-32a-FIPV-N was constructed in vitro and then transformed into E.coli T7 shuffle competent cells for prokaryotic expression,and the highly soluble FIPV-N protein was expressed as an antigen to immunize experimental rabbits.Antibody titer was determined after four times of immunization.the spleen RNA was extracted when the titer reached 1.2×10⁵,and the c DNA was obtained by reverse transcription afterwards.Then the target genes of the variable region of the heavy chain and the variable region of the light chain of the antibody were amplified,and then spliced into sc Fv by SOE-PCR.The sc Fv target fragment was cloned into the vector p Comb3XSS to form the p Comb3XSS-sc Fv expressing plasmid.Afterwards the plasmid was electrotransfected into ER2738 competent cells,which was infected with M13K07 helper phage to construct a rabbit-derived phage single-chain antibody library with a capacity of 3×10⁸ cfu/m L.The N protein was used as the target protein for panning.After 2 rounds of enrichment screening,11 strong positive（P/N>3）sc Fv strains were identified by phage-ELISA.The sequencing results showed that3 specific sc Fv strains were successfully screened.A sc Fv strain with successful sequencing results was selected and cloned into the eukaryotic vector p TT5-Human-Fc,which was transfected into HEK 293F cell line for eukaryotic expression afterwards.Protein A was used for purification to obtain recombinant Fc-sc Fv-N antibody.The affinity was detected by ELISA,and EC₅₀ was 7.938 ng/m L,indicating that the recombinant antibody has activity and good affinity.The specific sc Fv panned out in this study can be further studied for its inhibitory effect on FIPV infection,and lay a foundation for studying its clinical application prospects.