Expression Of Recombinant ETEC Enterotoxin Protein LTA192-STa13and The Humoral Immune In Mice Induced By It

Calf diarrhea is a digestive disease caused by Enterotoxigenic E. coli(ETEC), which main feature is acute watery diarrhea, dehydration, autointoxication and heart failure in1-month-old calves. It is one of the main reasons which causes newborn calves stunted growth and death. The main virulence factor of ETEC is fimbriae and enterotoxin. Fimbriae plays a major role in the colonization of the small intestinal mucosa, which has strong adhesion. Then the bacteria produce enterotoxin to cause disease. Due to the continuous emergence of resistant strains of E. coli, the effect of drug treatment is getting worse. Therefore, the development of effective prevention of calf diarrhea in E. coli vaccine has important practical implications for the prevention of occurrence of the disease.Enterotoxin includes LT and STa, causing intestinal secretion dysfunction, leading to diarrhea. The purpose of this study was to explore the expression of mustational eltA and estA fusion coding gene, getting attenuated recombinant E. coli enterotoxin protein, inducing the humoral immunity ability with it, providing scientific data for the development of experimental E. coli diarrhea in calves vaccine.Expression of recombinant E. coli enterotoxin proteins. In this study, we got eltA and estA gene through PCR, mutated the eltA gene for a LTA192(Râ†'G) toxoid and the porcine estA gene for STa13(Gâ†'Q) toxoid, used LTA192as an adjuvant to carry the STa13toxoid for LTA192-STa13fusion antigens. And we inserted the genetic fusions of eltA-stA into the pMD-18T simple Vector. After sequence analysis, the fusion gene eltA-stA inserted into the expression vector pHUE and transformed into Rosetta to constuct prokaryotic expression system of Rosetta/pHUE-eltA-stA. The result of SDS-PAGE demonstrated that molecular weight of expressed recombinant protein LTA192-STa13was36Ku, which expressed in the form of inclusion bodies. The recombinant protein was16.8%of the total bacterial protein. The Western blot declared that expressed recombinant proteins LTA192-STa13occured specific reaction with His and STa monoclonal antibodies separately.The research of humoral immunity in mice induced by recombinant protein. The6-week-old kunming mices were randomly divided into three groups (Group A, B, C),5for each group of normal mices and pregnant mices. Group A, B and C were immunized intraperitoneally with the LTA192-STa13fusion antigens, inactivated recombinant E.coli Rosetta/pHUE-LTA192-STa13and physiological saline thrice every two weeks, at an interval of14days.The blood of normal mices, pregnancy mices and offsprings were collected to detect the antibody regularly. ELISA results showed that group A and B could stimulate mice to develop high titers of anti-LTA and anti-STa antibodies. The titers of group A against LTA and STa was1:68200,1:8960separately at14days after the third immunization. The titers of group B against LTA and STa was1:14900,1:3840separately at14days after the third immunization. The titers of pregnancy mices against LTA and STa was1:40228,1:8530separately after7days of childbirth in group A. The titers of pregnancy mices against LTA and STa was1:18285,1:3840separately after7days of childbirth in group B. The serum antibodies could be passed to offsprings through the foremilk. The titers of anti-LTA and anti-STa antibodies of group A reached1:3800and1:850at30days after birth. The titers of anti-LTA and anti-STa antibodies of group B reached1:1060and1:330. The titers of mices and pups in group A were significantly higher than group B.14days after the third immunization, mices were injected intraperitonealiy by toxin of10LD50ETEC (LT+). Neutralization test in vivo showed the mices of group A and B could be protected, in which the group of LTA192-STa13fusion protein mices were all healthy and living, the control group died. Suckling mice lavage experiment showed that the serum from the group of recombinant protein mices and inactivated E.coli can neutralize STa. The mices were healthy and living without the emergence of intestinal fluid. The control group were all dead.The serum from mices was used to neutralize the virulence of LT to Y-1cell after third immunization with neutralizing titers tested. The result demonstrated that the higher neutralizing titers of recombinant protein group was89.13,1:22.59for the group of inactivated E.coli.This study showed that the recombinant LTA192-STa13protein had a good immunogenicity, induced immunized mices to generate a good humoral immune response, which can be candidate biological agents for prevention of E. coli diarrhea in calves.