| Swine E. coli disease,with various serotypes such as k88(F4), k99(F5), 987P(F6), F41,O138, O139 and O141, is an important disease caused by Enterotoxigenic Eseheriehia coli(ETEC), etc., which mainly affects weaned and newborn piglets. Currently, the prevention and control of ETEC include antibiotic treatment, multivalent vaccine, hyperimmune serum, etc., but either the measures aim to certain(or a few) serotypes, making it difficult to prevent and control multiple serotypes simultaneously.In previous studies, three broad-spectrum target antigens with good immunogenicity were selected from deleted in type II secretion system ETEC strains via reverse vaccinology. The mice were vaccinated with immunogen combining three broad-spectrum by different routes, and then challenged with multiple serotypes(serotype F4). The serum IgG and fecal IgA were detected, and protection rate calculated after challenge to evaluate the effectiveness of immunization, for preventing multiple serotypes and laying the foundation of universal vaccine against ETEC in swine. Simultaneously,double indirect ELISA was constructed with F4-F5 as target which may provide an effective mean for the serological and epidemiological analysis of ETEC. Thses results are as follows:(1) Establishment of indirect ELISA using F4-F5 fusion protein:Based on GenBank, the conserved domains of F4 and F5 genes were selected to optimize their codons,synthesized and then cloned into prokaryotic expression vector to express recombinant protein, His-F4-F5. The purified His-F4-F5 was used as the target protein to detect ETEC antibody.Using Restriction endonuclease digestion and DNA sequence analysis the results of the recombinant strain BL21(pColdI-F4-F5) was successfully constructed, and SDS-PAGE and Western blot assay showed that fusion protein F4-F5 of 29 kD was expressed and could react with positive hyperimmune serum of ETEC specifically with a single strip. The opitimal conditions of indirect ELISA were as follows: the concentration of coating antigen was 6 ng/well;the dilution of test serum and HRP-labeled goat anti-pig IgG were 1:200 and 1:15000, respectively. Which were Blocked with5% skimmed milk for 2h and incubated for 15 min. The F4-F5 indirect ELISA established in this study is characterited by high specificity, reproducibility and stability.(2) Preparation of universal immunogen and mice experiment: Three proteins of Escherichia coli were expressed in prokaryotic expression system,named as 734,739 and 696. The proteins were mixed at a ratio of 1: 1: 1, then mixed and emulsified with white oil adjuvant, wateradjuvant(gel 01) and mucosal immune adjuvant(LTMT),respectively, to prepare the vaccines.The mice were inoculated with the vaccines subcutaneously and intraperitoneally. After the second immunization, the mice were challenged with 5MLD(minimum lethal dose) F4. The serums and faces were collected before and after immunization to detect IgG and sIgA by indirect ELISA! After challenge, the mental state were observed and the dead number were recorded. The intestinal lavage sIgA of all survived mice were detected seven days later.The results showed: in the subcutaneous immunization group, 739 + 734 + 696 + gel 01(IV) and739 + 734 + 696 + LTMT + white oil(V) induced high titers of serum IgG, while lower fecal and intestinal IgA.In groupVthe, IgA titers were significantly higher than that in group IV,indicating that LTMT of subcutaneous immunization was still able to induce mucosal antibody sIgA. However, the protection rate(20%) of group V was significantly lower than group IV(80%) which needs to be analyzed in future. In intraperitoneal immunization group, 696 + 739 +734 + LTMT, compared to the subcutaneous immunization group, 739 + 734 + 696 + LTMT(III), induced higher serum IgG with similar feces and intestinal sIgA titers, indicating that LTMT could induce sIgA by subcutaneous and intraperitoneal inoculation. The protection rate between the two groups showed a big difference,with the abdominal cavity group(80%)significantly higher than subcutaneous III group(60%). Through comprehensive analysis, our preliminary conclusion is that either subcutaneous or intraperitoneal,739 + 734 + 696 + LTMT is a good multivalent immunogen; 739 + 734 + 696 and water adjuvant gel 01 emulsion vaccine are also good multivalent immunogen by subcutaneous immunization. |
