Study On The Construction And Expression Of Lactococcus Lactis Expression Vector Of PEDV COE Gene

Porcine epidemic diarrhea virus(PEDV) belong to Coronviridae,Coronavirus.PEDV is the etiological of porcine epidemic diarrhea(PED),which is a highly contagious enteric disease in pigs causing vomit, diarrhea,dehydration.For the tremendous economical loss and weak immunoprotection effect of inactivated vaccine,the research on genetically engineering vaccine of PEDV has a wide implemented perspective.The spike(S) glycoprotein of PEDV is the predominant inducer of neutralization antibody.The partial S glycoprotein of PEDV encoded by COE gene also is the inducer of neutralization antibody and has the antigenicity against PEDV.It has been identified that,the immunoprotection effect of IgG,IgM in blood serum wasn't good,but the antibody(sIgA) induced by enter-membrane immune was effective.For the above,select a carrier system that safe,could multiply in intestine is important to the protecting of PED.In this study,Lactococcus lactis NZ3900 was selected as receptor strain to express COE gene,which combined antibiosis function with specific immunity together.The aim was to lay foundation for developing the gene engineering Lactococcus lactis oral vaccine of PEDV.Lactococcus lactis NZ3900 with a deletion of lacF gene,combined with expression vector pNZ8149 that carrying lacF gene,use lactose as a selective marker,is a real eatable exogenous gene expression system.In this study,the partial S glycoprotein gene of porcine epidemic diarrhea virus(PEDV) was amplified by RT-PCR and sequenced.The gene consisted of 531bp and shared 99.4%nucleotide homology with PEDV CV777 strain.The COE gene(about 501 bp) in S glycoprotein gene was subcloned into the Lactococcus lactis vectors pNZ8149 and transformed into Lactococcus lactis NZ3900 by electroporation.The recombinant protein was detected by Western blot and IFA experiments after the bacteria induced by 1ng/mL nisin.The result indicated that the molecular weight of the expressed recombinant protein was about 20 kDa;the protein had the reactionogenicity with antibody against PEDV as expected;the protein was secreted and located on the surface of the bacteria.The result showed that we successfully constructed the epression rcombinants for PEDV COE gene in Lactococcus Lactis.Furthermore,the recombinant protein was located on the surface of the bacteria and had the reactionogenicity with antibody against PEDV.The study is expected to lay the foundation studies on the gene engineering Lactococcus lactis oral vaccine in their prevention of PED.