| Foot-and-Mouth disease (FMD), which is caused by foot-and-mouth disease virus(FMDV), is a highly contagious disease that seriously imperil the development of world livestock industry and international import and export trade. We have fully recognized that vaccination is a major method to prevent, control and even to eradicate the disease during the long struggle with the FMD. Although currently the conventional inactivated vaccine can be used in the world, the disease still affects millions of animals around the world and remains the main sanitary barrier to the commerce of animals and animal products. This at least in part due to the critical shortfalls of current inactivated antigen vaccines. Therefore, it is necessary to develop a novel FMD vaccine with more safety and efficiency. Epitope vaccine is a important vaccine style possessing great application and development prospects among numerous genetically engineering vaccine of FMDV.VP1 is a major antigenic protein of foot-and-mouth disease virus (FMDV), which induces the immune response against FMDV infection, and contains several epitopes of the virus. In this study, we designed and chemically synthesized a DNA fragment which encoding a tandem repeat protein of 136?160aa and 198?211aa of a strain of type Asia I FMDV VP1 gene, and cloned the gene of heavy chain constant region of sheep IgG. By using the BamH I, EcoR I and Xho I sites, both genes were cloned into pPROExHTb vector in turn to form a recombinant plasmid pPRO-FshIgG. A chimeric protein, named FshIgG, was obtained after transforming the pPRO-FshIgG into Escherichia coli BL21 (DE3) host cell and induced by IPTG. Meanwhile, we cloned the FMDV nonstructural protein 3D gene and realized the expression of the gene in Escherichia coli BL21 (DE3) host cell with denaturing condition and native condition.Inoculation with a vaccine containing 100μg FshIgG and a vaccine composed by 100μg FshIgG and 100μg denaturing 3D protein induced strong neutralizing antibody response in guinea pigs, and the inoculated guinea pigs were protected against 200 ID50 FMDV challenge. But the vaccine composed by 100μg FshIgG and 100μg native 3D protein induced significantly lower neutralizing antibody level than above two groupes. Further study of T-cell proliferation assays in guinea pigs showed that native 3D protein can inhabit the immune response to the FshIgG protein by depressing the proliferation ability of lymphocytes, this lead to the immunized guinea pigs can't be protected when challenged using 200 ID50 FMDV. Otherwise, inoculation with the vaccine containing 100μg FshIgG or vaccine composed by 100μg FshIgG and 100μg denaturing 3D protein also induced high level antibody in sheep, and the neutralizing antibody titre of the latter is higher than the former. According to the theory of the relationship between the protectivity with neutralizing antibody titre determined by liquid-phase blocking enzyme-linked immunosorbent assay, it can be deduced that all the sheep vaccinated by the two vaccine can be protected from natural infection.Thereby, our study indicated that the heavy chain constant region of sheep IgG can act as the carrier protein for FMDV peptide epitopes, and FshIgG is a potential multiepitope peptide vaccine candidate to prevent FMDV infection, and the combination of FshIgG and denaturing 3D protein is a good compatibility method. |
