Study On Recombinant Rabies Vaccine With G-LTB Carried By Attenuated Salmonella And Oral Immunization In Mice

G gene of rabies virus can be expressed into 67kd,524 amino acids glycoprotein. The protein can only induce neutralizing antibodies and protective immune response in the major structural protein, its immune function equals to whole virus vaccine. Extracellular domain takes responsibility for rabies virus glycoprotein antigenicity, tissue tropism and virulence. It is not only an effective protective antigen, but can also induce neutralizing antibodies and stimulate cellular immunity.Escherichia coli heat-labile enterotoxin (LT) consists of one A subunit and one B subunit by five non-covalently linked to form the pentamer. Studies showed that LTB is a good mucosal adjuvant with strong immunomodulatory effects. When LTB costimulates organism combined with other antigens, it may promote the body to produce specific antibodies against the costimulatory antigens and shows adjuvant activity.In recent years, many studies have proved that Salmonella typhimurium can be used as the ideal vaccine carrier of bacteria, viruses, parasites and tumors antigen. they can carry foreign genes to highly express protein and fully stimulate the humoral and cellular immunity. Therefore, the carrier based on Salmonella typhimurium in the field of vaccine research attracts much attention of many scholars at home and abroad.In this study, we had an analysis of bioinformatics on RV G gene and LTB gene by bioinformatics software. According to the published sequence of GenBank, two pairs primers were designed by Oligo 6. The pMD-G and pMD-LTB were used as templates to amplify G-linker- and -linker-LTB fragment by PCR. Gene splicing by overlap extension (SOE) PCR was executed to amplify the product G-LTB fusion gene, and then the fusion gene was combined to pMD18-T cloning vector by T-A ligation reaction. After pMD-G-LTB, pVAX1 and pVAX1-ori were digested by BamHâ… and Xhoâ… respectively, the obtained G-LTB fusion gene was ligased to the vector by T4 DNA ligase. Identification by PCR and digestion showed the results were correct and sequencing identification showed that the nucleotide sequence variation of foreign gene did not occur, in conformity with gene sequence published on the GenBank. The recombinant plasmids pVAX1-G-LTB and pVAXl-ori-G-LTB were transformed into E.coli JM109, and then plasmids were extracted and transfected into BHK-21 cells. After 48h cultured, immunofluorescence analysis showed that G-LTB fusion protein was successfully expressed in BHK-21. About 70kd target band was detected by western blot and demonstrated these recombinant eukaryotic expression plasmids pVAXl-G-LTB and pVAXl-ori-G-LTB were successfully constructed in the study.To further test the immunogenicity of G-LTB fusion gene, the plasmids pVAXl, pVAX1-G, pVAXl-G-LTB and pVAXl-ori-G-LTB were electroporated into attenuated Salmonella typhimurium mutant strain phoP/phoQ. The recombinants strains were used to orally immune 6-8 week female BALB/C mice at 1010CFU, immunization twice per 10 days. IgG antibodies test showed that IgG antibodies level in the phoP/phoQ(pVAX1-G-LTB) and phoP/phoQ(pVAX1-ori-G-LTB) immunization groups were significant higher than the controls group. Neutralizing antibodies test showed the antibodies level were more than 0.5IU/mL in two immunization groups, Conversely results were in control groups and the PBS group. The animal protection test showed that phoP/phoQ(pVAXl-G-LTB) and phoP/phoQ(pVAXl-ori-G-LTB) groups gained 50% and 60% of protection respectively and their IL-2 test also showed statistical significance compared with control group. In summary, we can safety draw a conclusion that recombinant rabies vaccine with G-LTB carried by attenuated Salmonella could induce humoral and cellular immune responses in againsting rabies virus.Compared with intramuscular or gene gun immunization, oral vaccine with saving time, convenient, inexpensive, easy to herd immunity, is a new way to develop low-cost practical oral DNA vaccine. Therefore, this study will lay a solid foundation for further development of the novel mucosal oral vaccine against rabies.