| Bovine Asiaâ… Foot-and-mouth disease (FMD) is a highly contagious viral disease caused by the infection of Bovine Asiaâ… Foot-and-mouth disease virus (FMDV).Inoculation with a chemically inactivated Bovine Asiaâ… FMDV vaccine is currently used to control the disease. The vaccines, although generally effective, do have some disadvantages. One is the possible escape of live virus from vaccine production. The other is the incomplete inactivation of the virus may cause the outbreak of FMD, as reported in Europe for several times. For the rational, we have tried to develop a recombinant FMDV vaccine consisting of the major B cell epitope and the T cell epitope from VP1 protein that is the key structure in FMDV for inducing the production of neutralizing antibodies.To develop a recombinant vaccine of bovine Asiaâ… FMDV with high efficacy, we have constructed a gene fragment (HBB)2 encoding for the vaccine. The gene fragment consists of four B gene fragments and two H gene fragments. The B gene fragment is composed of the encoding gene of a B epitope and a T epitope from VP1 protein and restriction enzyme cutting sites of EcoRâ… , Hindâ…¢, Bglâ…¡and BamHâ… on its two ends. The H gene fragment contains the same restriction enzyme cutting sites as the B gene fragment, but their B epitope and T epitope are different. Then, the (HBB)2 gene was subcloned into two vectors respectively to express two recombinant vaccines of bovine Asiaâ… FMDV in E.coli.This thesis includes the following parts.1. The construction of bovine Asiaâ… FMDV VP1 epitope hetero6 (HBB)2 Firstly, we constructed the BB gene using isocaudarner. The B gene in pMD18T-B and the H gene in pMD18T-H (provided by Haifei Xu and Zhao Cao in our lab) were released by Bglâ…¡/BamH digesting and recovered from agerose gel, respectively. pMD18T-B was digested by Bglâ…¡to get a linear vector. The B gene was cloned into the upstream of the B gene in pMD18T-B, obtaining pMD18T-BB. Then, the H gene was cloned into the upstream of BB gene in pMD18T-BB, constructing pMD18T-HBB. Finally, pMD18T-(HBB)2, containing the recombinant vaccine encoding gene, was constructed by cloning HBB gene into the upstream of HBB gene in pMD18T-HBB.2. The expression of recombinant vaccine of bovine Asiaâ… FMDVThe (HBB)2 gene was subcloned into pET28a and secretary vector pET26b, respectively. pET28a-(HBB)2 and pET26b-(HBB)2 were transformed into expression host BL21 (DE3), respectively. Positive clones were selected and inoculated into medium. After being induced in the presence of isopropyl-beta-d-thiogalactopyranoside (IPTG), the bacteria were collected and lysed. The lysate was analyzed by SDS-PAGE, revealing the high level-expressed proteins that were confirmed by Western blot.The recombinant protein expressed in BL21 by pET28a-(HBB)2 was designated as A4a, and that expressed by pET26b-(HBB)2 was designated as A4b. The A4a, expressing in the endochylema of BL21, was totally inclusion body, whereas the A4b was partly secreted into the pericellular space of BL21.3. The optimization of A4b expressionDuring the purification of A4a from inclusion bodies, denaturation and renaturation might affect the correct protein folding and biological activity. However, the purification of A4b from pericellular space did not require denaturation and renaturation. To increase the secretary amount of A4b, we optimized the expression conditions, such as prolonging the induction time, decreasing the inductor concentration and induction temperature. The results showed that the secretary amount of A4b was not increased by these measures.4. The study on A4aAfter purified by nickel affinity chromatograph and desalinated by gel filtration chromatograph, the purity of A4a was more than 90%. The stability analysis of A4a showed that the A4a was stable in low temperature. To investigate the immunogenecity of A4a, guinea pig immunization was carried out. A4a were combined with 206 oil adjuvant to immunize guinea pigs on day 0 and day 22. Sera were collected on day 0, 7, 21, 29, 36, 43, 50 and 64. The specific antibody level in serum was detected by indirect ELISA method in which the inactived bovine Asiaâ… FMDV was used as coating antigen. To confirm the protective effect of sera, FMDV neutralizing antibodies in sera were detected by cell protective assays and suckling mice protective assays.The result of ELISA showed that the A4a could stimulate the guinea pig to product high level of antibody specific to bovine Asiaâ… FMDV. The antibody titer reached its peak value on day 29, similar to the inactived FMDV vaccine.The cell protective assays showed that the titer of neutralizing antibody in the sera reached 1/724 on day 43, and was higher than that of the inactived FMDV vaccine. The sera on day 21 were also examined by the suckling mice protection assays. The result was as followed: 1/32 diluted sera provided total protection of the suckling mice, and 1/64 diluted sera provided 3/4 protection.In this study, a gene encoding for the vaccine of bovine Asiaâ… FMDV was constructed and subcloned into pET28a and pET26b, respectively. Two recombinant proteins, A4a and A4b, were respectively expressed by pET28a-(HBB)2 and pET26b-(HBB)2 in E.coli. The expression of secretary A4b was too low to satisfy the purification and immunization, even though we optimized the expression conditions. Althogh treated by denaturation and renaturation in purification, the A4a can induce high titer of neutralizing antibody against bovine Asiaâ… FMDV. |
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