| Echinococcosis (Hydatidosis) is a worldwide major zoonoses caused by infection with larval stage of Echinococcus granulosus(Eg) in mammal, which are harmful to both public health and animal husbandry. Immunization with vaccine is the most effective way to prevent and control this disease. A Sheep echinococcosis EG95 genetic engineering subunit vaccine has been proved to be effective. However, the expression of EG95 protein in the recombinant vaccine is low and not soluble, which restricts the large-scale production and application of this vaccine.In order to improve the expression efficiency and soluble abilities of the proteins, we modified EG95 to EG95s, and optimized the expression conditions, then developed an indirect ELISA for detection of EG95 antibodies, moreover, studied the immunogenicity of the EG95s recombinant proteins.According to the solubility and antigenicity of the complete sequence of EG95 gene, the highly hydrophilic epitope region in EG95 gene, named as EG95s, was cloned, and 1-3 copies of EG95s (348bp) were tandemly insertes into pET-32a by isoschizomer construction method. Finally, the recombinant plasmids were transformed into BL21(DE3) for expression. The expressed fusion proteins were accordingly named as HIS-1EG95s, HIS-2EG95s and HIS-3EG95s. The results showed that the expressed proteins were high productivity with good solubility, and easy to purify, moreover the percentage of the target protein in the total proteins were increased, which is benefit for further development and utilization of the vaccine.The expression conditions of pET-32a-3EG95s were optimized in 100 mL shakingflask, including different host bacterias, cultivation culture condition and induction condition, and the results were orthorgonally analyzed. The results indicated that the OD600 value of the recombinant bacteria (pET-32a-3EG95s/ Rosseta (DE3) culture could reach 7.65, which means the culture density was increased 1.77-fold than before, and the protein production was also increased 2.64-fold.Using purified HIS-1EG95s as coating antigen, an indirect ELISA for the detection of antibody against EG95 in serum was developed (0.001μg/μL of the protein for coating, the dilution of negative and positive sera for control was 1:200, and the working concentration of HRP- IgG was 1:5000).This assay has good repeatability, sensitivity and pecificity, which could be applied for monitoring the antibody level of immunized animal. So, it afforded a suite means to assess immunogenicity of EG95s recombinant protein.The immunogenicities of the purified rencombinat proteins, HIS-1EG95s, HIS-2EG95s and HIS-3EG95s, were further analyed by Western-blot. The results showed that all the recombinant proteins produced strong staining with positive sera. BALB/c mice were vaccinated with these purified rencombinat proteins at the same dose and procedure respectively, then detected the levels of specific antibodies at different stage. The result indicated that all the three proteins have good immunogenicities, among of them, HIS-3EG95s was the best one to stimulate mice to generate specific antibodies. In general, the present results indicate that the recombinant proteins were successfully and efficiently expressed. An indirect ELISA for the detection of antibody against EG95 in serum was developed as a matching detection method for the EG95 genetic engineering subunit vaccine. All the recombinant proteins have good immunogenieities, and HIS-3EG95s, which contains more interest protein could has the best ability of inducing specificy antibody. Our data provide basis and proofs for the further development of the production of Echinococcus granulosus vaccine.
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