Expression Of Type A Foot-and-mouth Disease Virus VP1 Gene In Attenuated Salmonella Typhimurium Expression Vector And Protection Of Oral Immunization In Mice

【Objective】Foot-and-mouth disease(FMD) is an acute, febrile and highly contagious viral disease which is caused by Foot-and-mouth disease virus(FMDV), mainly infected the domestic and wild artiodactyls. It leads the artiodactyls to blister on the hoof, mouth and breast, and associates with symptoms of fever and sore. It results in severe economic loss for livestock industries and in great national economic loss. It was enlisted as members on the List A of contagious diseases by OIE. A basis constructed and expressed by type A FMDV VP1 gene provides a feasibility of using attenuated Salmonella typhimurium as vector for an oral immunization.【Method】(1)After FMDV VP1 gene was amplified and sequenced, the gene sequence was optimized and synthesized according to the codon bias of attenuated Salmonella typhimurium, and inserted into the expression plasmid p YA3341. The recombinant plasmid was transformed into E.coli X6212 for amplification and identification by PCR and restriction enzyme digestion. The recombinant plasmid p YA3341-VP1 was further transformed into the intermediate host bacteria of attenuated Salmonella typhimurium X3730, and then the plasmid extracted from X3730 was transformed into the expression bacteria X4550 for protein expression.The protein expression was tested by SDS-PAGE and Western-blot after adding IPTG at hour 3.5 cultivation.( 2) After the BALB/c mice were immunized by oral route with recombinant attenuated Salmonella typhimurium X4550(p YA3341-VP1) in twice, VP1-specific antibodies were monitored by ELISA at 0, 28, 42,and 56 days.【Result】(1)Results showed that VP1 protein of 23 ku was successfully expressed in recombinant attenuated Salmonella typhimurium X4550(p YA3341-VP1).(2)The Ig G in serum of recombinant attenuated Salmonella typhimurium X4550(p YA3341-VP1) was higher than control group(PBS group and Empty plasmid group).Compared with inactivated vaccines group, the Ig G in serum were undesirable. The Ig A occurred early in serum and disappeared quickly. In addition, recombinant attenuated Salmonella typhimurium X4550(p YA3341-VP1) group reduced the s Ig A in mucosa of mice, it showed that recombinant bacteria could stimulate both mucosal and systemic immunity, in the meanwhile, the s Ig A of the control group(PBS group and inactivated vaccines group) has not increased. The reason for the s Ig A of empty plasmid group has increased probably is immunogenicity doesn’t delete completely, yet the result needs more experiments to prove. In the Virus neutralization test, sera from mice immunized with recombinant attenuated Salmonella typhimurium X4550(p YA3341-VP1) were significantly higher than negative control groups. Sera from mice treated with X4550(p YA3341) or PBS showed no neutralizing antibody activity.【Conclusion】 We immune mice by oral vaccination in this study.Using attenuated Salmonella typhimurium as a vector to present antigens not only induce humoral immune response and cell mediated immune responses but also stimulate mucosal immunity. Although the Ig G level is effective than the inactivated vaccines, musocal vaccine by oral route is safer, cheaper and has adjuvanteffects; also, it is competitive as FMD netype of vaccine.