Construction And Immune Efficacy Evaluation Of The Recombinant Lactobacillus Casei Surface-displayed 987P And K88 Of ETEC

Piglet yellow diarrhea caused by Enterotoxigenic Escherichia coli(ETEC)is a common and frequently-occurring disease in intensive pig rearing.It is a highly lethal infectious disease that causes diarrhea in newborn piglets,which has caused the pig industry Huge economic loss.ETEC has two pathogenic factors: Adhesin and enterotoxin.After ETEC invades the body,it first colonizes the intestine with the adhesion of Adhesin,reproduces continuously,and then produces enterotoxin,which causes diarrhea.Therefore,Adhesin is the first cause of ETEC disease.The step played a decisive role.The Adhesin often carried by ETEC is K88,K99,987 P,F41.The recombinant Lactobacillus casei(L.casei)expressing the 987 P and K88 protein of ETEC was constructed,and the immunogenicity of the recombinant L.casei was analyzed.According to the gene sequences of ETEC 987 P and ETEC K88 in Gene Bank,the specific primers were designed and synthesized for getting the insert genes.A prokaryotic expression system expressing 987 P protein and K88 protein respectively were constructed.Western blotting verification results showed that the recombinant protein had of good reactivity and could be used for subsequent experiments.In this experiment,the 987 P and K88 genes were sequentially connected to the shuttle plasmid p LA.The ligation system was transformed into Escherichia coli BL21 competent cells.The positive recombinant plasmids were electron-transformed into L.casei for the recombinant bacteria identified as positive by PCR.Western blotting detected the recombinant protein with a size of about 85 k Da.The positive expressing bacteria were screened by indirect immunofluorescence and tested by flow cytometry.The positive rate of 10,000 recombinant bacteria reached 92.81%.The laser confocal detection showed that the protein was successfully displayed on the surface of the recombinant bacteria.The recombinant L.casei was named p LA-987P-K88/L.casei.In order to explore the immune effect of the recombinant p LA-987P-K88/L.casei,5-6weeks SPF BALB/c mice were intragastrically administrated by the recombinant bacteria for three times.The groups were designed as p LA-987P-K88/L.casei group,p LA-987P/L.casei group,p LA-K88/L.casei group,p LA/L.casei group,saline group and blank control group.The indirect ELISA method was used to detect Ig G antibody titers in serum and s Ig A antibody titers in feces,intestines,and lungs.The mouse were challenged by the strains ETEC K88 and 987 P,respectively,to evaluate the protection rate of mice in the test group.The results showed that the antibody levels of Ig G and s Ig A in each immunization group were significantly higher than those in the control group(p<0.0001).There were no significant differences in antibody levels between the p LA-987P-K88/L.casei group and the p LA-987P/L.casei group and p LA-K88/L.casei group.The challenge test was carried out on days 14 after the final immunization.The protection rates for mice in the p LA-987P-K88/L.casei group against the reference strains enterotoxigenic E.coli C83916(987P)and C83902(K88)were 80% and 90%,respectively.The lymphocyte proliferation test showed that the level of cellular immunity was significantly increased after immunization with the recombinant bacteria,which was significantly higher than that in the p LA/L.casei group(p<0.001).Therefore,the above results indicated that the p LA-987P-K88/L.casei induced the mucosal immune response,cellular immune response,and humoral immune response,suggesting that p LA-987P-K88/L.casei effectively prevented the mouse from ETEC infection.Compared with immunizing with p LA-987P/L.casei and p LA-K88/L.casei,it has more economic benefits.The candidate vaccine of the multivalent recombinant lactic acid bacteria provides an important research basis for the development of oral vaccines against the diarrhea disease.