Cloning And Prokaryotic Expression Of Xinjiang Karakul Sheep Fas Gene And The Analysis Of Its Antigenicity

Fas gene is a member of TNFa receptor super family.It play a important role in keeping the balance of the immune cells.The research will lay a foundation for the animal’s pathology and disease diagnostics through cloning the Fas gene and constructing of prokaryotic expression vector of the Fas gene.Specific primers for Fas (TNF receptor superfamily, member6) were designed through GenBank database,the protective bases and the EcoR I and Xho I restriction enzyme sites were added at the ends of the primer.Total RNA was extracted from peripheral blood lymphocyte (PBL) of karakul sheep by TRIzol reagent, than a specific sequence was amplified by reverse transcription-polymerase chain reaction(RT-PCR).The sequence was ligated to pMD-18T vector after purified.the recombinant sequence was identified by double digests method and PCR.The results suggest that the fragment contained994base paris and the amino acid sequence was analyzed using the online software.The results suggest that the structure and function of Fas gene possessed three typical functional domain including two members of TNFR super family.an trans membrane domain and a C-terminal death domain.Besides.two potential N-glycosylation sites,eleven potential phosphory sites and two main hydrophilic in Fas amino acid sequence were found,but no O-glycosylation sites.The main secondary structure of protein was composed of a-helic and randon coil.The Fas gene was linked into prokaryotic expression vector pET28a and the recombinant fusion expression plasmid pET28a-Fas was constructed. Then pET28a-Fas was transformed into E.coliBL21(DE3).The different concentration of IPTG and different temperature was set up.and the optimal conditions was defined,then the recombinant fusion protein was purified.The results showed that the insert direction sand reading frame of Fas gene were right.The pET28a-Fas gene had expressed in E.coliBL21.The expression capacity pET28a-Fas was15.6%by optimizing expressed condition,and the MW of the fusion protein was39.7KDa.Through the Agar Immunodiffusion Test and Western blot,the result showed that the Fas have immunogenicity and reactionogenicity.