Construction Of A Phage Display Antibody Library And Selection Of Special ScFv Against The Merozoite Of E. Acervulina

Coccidiosis coused by Eimeria is a very several globally psrasitosis to effect poultry .Now coccidiosis is still mainly controlled with medication .Owing to the repeated appearance of drug-resistant strains of coccidiosis ,the expensive costs of new anticoccidial drug being developed and the serious problems of drug residues in poultry production, the development of vaccine against coccidiosis becomes more important in recent years .Total RNA was extracted from the spleen cells of BALB/c mice. The frist chain of cDNA which derived from the total RNA with Oligo(dT)₁₅Primer by RT was used as template,VH and VL genes was amplified by PCR using gene specific primers. PCR pruduct be examined by agarose gel electrophoresis and purified by agarose gel DNA purificatin kit ,the result shows the genes of VH and VL amplified were about 420bp and 390bp respectively.And joined by a DNA linker encoding peptide as a single-chain Fv(ScFv)Fragment with SOE-PCR, ScFv was about 750bp and the Sfi I and Not I restriction site were inlet in it .PCR products were purified from gel then we can get the ScFv gene . The ScFv gene repertoires were overdigested by Sfi I and Not I separately . The ligation mixes of ScFv and PCANTAB-5E were transformed into E.coli TG1. Result showed that ScFv gene was correctly inserted into phagemid vector in frame.Those amplicillin resistant bacteria colonies were scraped into LB medium and superinfected by M13KO7 helper phage .After overnight induction in LB-AK medium containing no glucose ,we got recombinant ScFv phage .The phage antibody library was panned using the merozoite of Eimeria acervulina.After four rounds of panning , ELISA was performed to determine the specificity of the phage antibody.Strongly positive recombinant phage clones were used to infect log-phage E.coli HB2151.Expression of soluble ScFv was induced by adding IPTG and the cultures grown. The Mw of ScFv is about 32KD by SDS-PAGE.Mouse ScFv phage display library was constructed successfully and we got the special ScFv gene against the merozoite of Eimeria acervulina.