Establishment Of Anti-IBV Single-Chain Fragment Variable Prokaryotic Expression Plasmids And An Indirect ELISA Screening Method

Single-chain fragments variable (scFvs) contain the complete antigen binding site, which includes the variable heavy (VH) and variable light (VL) domains, of an antibody. The VH domain is linked to a VL domain by an introduced flexible polypeptide Linker. An scFv is capable of binding its target antigens with an affinity similar to that of the parent monoclonal antibody. China is a large chicken-farming country, all kinds of chicken diseases, especially viral diseases seriously affect the prospects of chicken industry, reduce the economic benefit and even cause huge economic losses. Therefore, it is of very importance to develop antiviral drugs which have ideal immunity effect, good safety and low cost. ScFvs are becoming popular therapeutic alternatives to full length monoclonal antibodies since they are smaller, most of them can be produced more economically and are easily amendable to genetic manipulation. Of the dozens of antibody fragments that have entered clinical studies at home and abroad, 35 percent are scFvs. It is one of the most rapidly developing recombinant antibodies. However, compared with human source scFvs, animal source scFvs are barely studied, and poultry scFvs research are even much rarer. This study used pOPE101-XP as vector, obtained anti-Avian Infectious Bronchitis Virus(IBV) scFvs, and established an indirect enzyme-linked immuno sorbent assay (ELISA) for anti-IBV scFv screening.1. Construction and prokaryotic expression of anti-IBV scFv.Chicken spleen mRNA was extracted after immunization by IBV H120. VH and VL genes were amplified by RT-PCR and PCR. ScFv gene was constructed by VH and VL chains via a Linker like VH-Linker-VL. The recombinant expression plasmid was constructed by inserting scFv into vector pOPE101-XP and was proved by sequencing. SDS-PAGE was used for analysis of the soluble scFv from periplasmic space after induced by IPTG. Sequencing result showed that anti-IBV scFv was successfully constructed. SDS-PAGE showed soluble scFv was successfully expressed.2. Amplification and purification of IBV-M41, and establishment of an indirect ELISA for anti-IBV scFv screening.IBV-M41 isolate was inoculated into 10-day SPF chicken embryos. The allantoic fluid (AAF) which was concentrated through dialysis bag was purified after ultracentrifugation and gradient centrifugation. The conclusion was identified by SDS-PAGE. Ultraviolet spectrophotometry was used to measure the concentration of IBV-M41. The concentration of anti-IBV scFv was detected by indirect ELISA.Purified IBV-M41 was used to immunize mice and coat ELISA plate in order to screen the specifically binding scFvs. An indirect ELISA screening method was established by optimizing the reaction condition: IBV-M41 was diluted with 20 fold (61.36μg/mL) as the coating antibody, coating in 4℃overnight; blocking in 37℃for 1 h by 2% skim milk powder; soluble scFv was diluted with 100 fold (142.7μg/mL), reacting in 37℃for 2 h; Myc-Tag Mouse mAb was diluted with 2000 fold, reacting in 37℃for 2 h; Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG was diluted with 4000 fold, reacting in 37℃for 1 h. The lower limit of detectability was 17.83μg/mL confirmed by the sensitivity test. The scFv only reacted with IBV confirmed by specificity test. Repetitive experiments (coefficient of variation was 2.56 %, 2.78 %) showed stability of the method.