Construction Of Lactic Acid Bacteria Vectors Fusion Expressing PgsA,CfaB And FliC Genes And Evaluation Of Immune Effect

Enterotoxigenic(ETEC)is one of the most important pathogens that cause porcine post weaning diarrhea(PWD)in piglets.ETEC fimbriae promote the attachment of bacteria to intestinal epithelial cells,and only after colonization of the intestine can they have the opportunity to release enterotoxin into the intestine.The CFA/I family of fimbriae is the main colonization factor antigens(CFAs),and it is also the fimbriae that covers the most serotypes of E.coli.CfaB protein is the main subunit protein of CFA/I,and it has been verified that it has excellent antigenicity and can induce the body to produce strong mucosal immunity.Salmonella is the pathogen of Salmonellosis that causes zoonotic diseases.Newborn animals carry this pathogen,and large animals carry and spread the bacteria asymptomatically throughout their lives.Studies have shown that the flagellin of Salmonella is weakly virulent and is a known antigen.It induces innate immune response through TLR5 and combines with other antigens to enhance immune response.The mixed infection of the two often aggravates the disease,and eventually the piglets with insufficiency of the intestinal environment die,which has always brought huge economic losses to the pig industry.Although antibacterial drugs have been used to treat these two kinds of bacteria,with the emergence of multi-drug resistant bacteria(MDROS),antibiotics lose their ability to treat,and safe and effective genetic engineering double vaccines are ideal prevention and control methods.At present,there is no report about the use of CfaB gene and FliC gene lactic acid bacteria dual genetic engineering vaccine.In this experiment,the fusion expression of Escherichia coli CfaB protein,truncated Salmonella flagellin FliC,and truncated Bacillus subtilis glutamate synthase pgsA'genes were synthesized and fused to the expression vector pNZ8048 using T₄ligase to obtain the recombinant Expression vector pNZ8048-pgsA'-CfaB,pNZ8048-pgsA'-FliC and pNZ8048-pgsA'-FliC-CfaB;then three recombinant plasmids were electroporated into Lactococcus lactis(Lactococcus lactis,L.lactis),and the recombinant was successfully constructed Lactococcus lactis NZ9000/pNZ8048-pgsA'-CfaB,NZ9000/pNZ8048-pgsA'-FliCand NZ9000/pNZ8048-pgsA'-FliC-CfaB;use Escherichia coli BL21 prokaryotic expression and purification of the proteins CfaB-His and FliC-His,and The polyclonal antibodies were prepared separately;Nisin was used to induce recombinant lactic acid bacteria to express the target protein,and the size of the target protein was determined by Western blot to be consistent with expectations.Mice were immunized with lactic acid bacteria orally,immunized 4 times,and mouse serum and small intestinal mucosa were collected at 10 days intervals.ELISA results showed that SIg A secreted by small intestinal mucosa of mice in CfaB,FliC and CfaB-FliC groups was significantly higher than that in PBS group(P<0.05)There was no significant difference in serum Ig G of mice in each group(P>0.05);the cytokine IL-6 in the serum of mice in the experimental group was significantly higher than that in the PBS group(P<0.05),but the levels in the serum of mice in each group There was no significant difference between TNF-?and IFN-?(P>0.05).In this study,a recombinant Lactococcus lactis expressing ETEC protein CfaB and Salmonella protein FliC was successfully constructed,which can induce obvious mucosal and cellular immune responses in mice,and is designed to develop a dual genetic engineering for the clinical prevention of porcine colibacillosis and porcine salmonellosis.The carrier vaccine lays the foundation.