Studies On Expression Of PRRSV ORF5with ETEC Fimbriae FaeG Fusion Gene In Pichia Pastoris

Porcine reproductive and respiratory syndrome (PRRS) is caused by porcine reproductiveand respiratory syndrome virus(PRRSV)is an acute,febrile,contagious disease with reproductivefailure in sows and respiratory disorder in piglets, which leads to great economic losses to thepig industry. Vaccination is the key measure to prevent and control the disease. However,inactivated vaccine and attenuated vaccine in the market is not protective completely, so it isvery important to develop the new vaccines. PRRSV GP5protein inducing immune response isthe main protective antigen. FaeG protein in fimbria of porcine enterotoxigenic E.coli(ETEC) isan adhesion factor and has strong adherence to mucous membrane receptors of intestinalepithelial cell, inducing the mucosal immune. The expression system of Pichia pastorisexpresses less protein with lower glycosylation by itself some of which needs anything toinduce. GP5and FaeG protein expressed in Pichia pastoris in the form of syncretic secretoryprotein in this study provides a new direction to genetic engineering vaccine.Pathological material inoculated with Marc-145cells until the typical CPE occurred. APRRSV strain identified by electron microscope observation and RT-PCR detection was namedJL-12. RT-PCR sequencing analysis shows: ORF7gene of JL-12has high similarity toAmerican PRRSV ORF7sequence and low similarity to the European Lelystad virus, whichproves JL-12strain belongs to the American type. ORF5and FaeG gene from PRRSV andETEC fimbriae were amplificated respectively by PCR and cloned into pMD-18T-Simple,which constructs two recombinant plasmid named pMD-ORF5and pMD-FaeG respectively.PRRSV ORF5was got from pMD-ORF5by double enzyme digestion and subcloned intopGAPZαA to construct the recombinant plasmid named pGAPZαA-ORF5. The FaeG gene wasgot by double enzyme digestion and subcloned into the pGAPZαA-ORF5to construct therecombinant plasmid named pGAPZαA-ORF5-FaeG. Then pGAPZαA-ORF5andpGAPZαA-ORF5-FaeG were transformed into Pichia pastoris expression system and whichobtain recombinant yeast of expresses secretory protein. The molecular mass of GP5protein andGP5-FaeG fusion protein were approximately24kDa,52kDa and both have immunogenicity ofPRRSV detected by Western-Blot.The two recombinant proteins were concentrated and inoculated mice in this study, thehumoral and cellular immune response index were detected. The results of specific IgG andsIgA showed that the group GP5protein and the group GP5-FaeG fusion protein both caninduce IgG and sIgA slightly, and the two groups have no significant difference in IgG. However, the content of slgA of the group GP5-FaeG fusion protein was significantly higherthan the group GP5protein. The two groups have no significant difference in lymphocytesrespectively induced by PRRSV and CoA and but have significant difference with the controlgroup. The content of INF-γ in lymphocyte was determined by ELISA, the results showed thatthe two groups both can produce limited content of INF-γ, but it has no significant difference.The results of lymphocyte proliferation assay and INF-γ shows that the recombinant proteinboth can stimulate the body to produce cellular immunity at a level.