Expression Of Recombinant E.coli Toxin Protein LT-IIc1B-STx2B-STa13 And The Humoral Immune In Mice Induced By It

Calf diarrhoea is caused by pathogenic Escherichia coli a common clinical disorders, mainly as diarrhea, dehydration, or sepsis, which is a major cause of death of newborn calves, bringing serious economic losses to the cattle industry. The main application of antibiotic treatment of E.coli disease is antibiotics,but the effect of the drug treatment is getting worse, Therefore, the development of an effective vaccine against Escherichia coli diarrhea in calveshas important practical implications for the prevention of occurrence of the disease.Virulence factors of calf’ diarrhea caused by E.coli are enterotoxin(LT,ST), Shiga toxin and adhesion.The enterotoxin includes heat-labile enterotoxin, LT and heat-stable enterotoxin, ST. Heat-labile enterotoxin can be divided into two types of LT-I and LT-II, Shiga toxin is divided into two types, type 1 and type 2, type 1 shiga toxin has weaker toxicity than type 2 shiga toxin. This study aims to obtain the recombinant proteins through chaining the STa 13 amino acid mutations gene and LT- IIc1 B subunit, Stx2 B subunit by the fusion of PCR technology together, as immunogen to study the induction of humoral immunity in mice, lay a experimental basis for the calves of E.coli vaccine research.In this study, we got LT-IIc1 B gene and Stx2 B gene respectively by PCR,connecting the STa13 gene through flexible connect and inserting the fusion gene to the pMD-18 T Simple,through digesting, connecting, transforming into Rossetta after sequence analysising to constuct prokaryotic expression system of Rossetta/p HUE- LT-IIc1B-Stx2B-STa13. The product of result by SDS-PAGE demonstrated that molecular weight of expressed recombinant protein LT-IIc1B-Stx2B-STa13 was 38 ku. The Western blot declared that expressed recombinant proteins LT-IIc1B-Stx2B-STa13 occured specific reaction with His and STa monoclonal antibodies separately.The 6-week-old kunming mices were randomly selected to divide into three groups(Group A, B,C),each group has 10 mices(5 for each group of normal mices and pregnant mices). Group A, B and C were immunized intraperitoneally with the LT-IIc1B-Stx2B-STa13 precipitation, the purified protein LT-IIc1B-Stx2B-STa13 and PBS, physiological saline thrice every two weeks, atan interval of 14 days.The blood of normal mices(A,B,C) were collected to detect the antibody regularly. ELISA results showed that group A and B could stimulate mice to develop high titers of anti-LT-IIc1, anti-Stx2 and anti-STa antibodies, The titers of group A against LT-IIc1 B, Stx2 B and STa was 1:15130, 1:10000,1:9744 separately at 14 days after the third immunization.. The titers of group B against LT-IIc1, Stx2 and STa was 1:11410,1:9017,1:8807 separately at 14 days after the third immunization. The titers of group C has not immune titer. Group A and group B pregnancy mice also produce higher serum antibody titer, two groups of rats at 30 d still have effective price,but group C did not produce serum antibody titer.14 days after the third immunization,mices were injected intraperitonealiy by toxin of 10LD50Stx2. Neutralization test in vivo showed the mices of group A and B could be protected, the control group C mice 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 diluted in times to use to neutralize the virulence of LT-II to Y-l cell and the virulence of Stx2 to Vero cell after third immunization with neutralizing titers tested. The result demonstrated that the higher neutralizing titers of recombinant protein group was1: 40.22, 1:20.11 for the group of inactivated E.coli.; The result of Stx2 to Vero cell after third immunization with neutralizing titers tested was 1:25.28(group A), 1:12.64(group B).This study showed that the recombinant protein LT-IIc1B-Stx2B-STa13 can lead to produce the humoral immune response in mice, the serum antibody of enterotoxin LT-IIc1, STa and shiga toxin Stx2 have the neutralization in vitro, The recombinant protein can be used as E.coli calf diarrhea vaccine candidate antigens.