Development Of Oral Rotavirus VP6 Subunit Vaccine By Plant Expression

By inserting the vp6 gene of human group A rotavirus into prokaryotic expression vector pGEX-KG, plasmid pGEX-VP6 was constructed and transformed into E.coli strain BL21 (DE3) . After IPTG induction, a fused protein of GST (glutathione S-transferase) with VP6 was expressed in E.Coli high level. By means of GST affinity chromatography and fast protein liquid chromatography (FPLC) , the VP6 protein was purified for preparation of a high titer polyclonal VP6-specific antibody in rabbit.For transient expression of the vp6 gene in Chenopodium amaranticolor by using Beet black scorch virus (BBSV) as a vector, the entire vp6 gene was cloned into a BBSV infectious cDNA as a substitute of the BBSV coat protein (cp)gene. The recombinant viral RNA was transcribed in vitro for mechanical inoculation of C. amaranticolor. Four to ten days post-inoculation, the expressions of VP6 mRNA and protein in the inoculated leaves were examined by RT-PCR or Western blotting, respectively, and further quantified by indirect ELISA.By using a crude recovery procedure and lyophilization, the total soluble proteins were extracted from both healthy and infected C. amaranticolor leaves for oral immunization of mice, in which three groups of female BALB/C mice were gavaged weekly with the protein preparations. In a period of 7 weeks following the first injection, the VP6-specific IgA liters in saliva, feces and intestinal mucosa of the mice, as well as the IgG in the serum, were detected by indirect or sandwich ELISA. The results showed that the mice immunized with the VP6 preparation from the infected leaves had high antibody titers, in contrast to that in the animals fed with healthy materials. In challenging infection by simian rotavirus SA-11, the severity and duration of diarrhea in passively immunized pups were reduced significantly, indicating that anti-VP6 antibodies generated in orally immunized female mice can be passed on to their pups for a heterotypic protection.Two plant expression vectors of pBVP6 and pBinBB-sVP6 both containing rotavirus vp6 gene coding for the intermediate coat protein were constructed for transformation of sugarbeet (Beta vulgaris L) mediated by Agrobacterium tumefaciens strain EHA101. The plantlets were regenarted on antibiotic selective media and the integration of the transgene was identified by PCR and Southern blotting.In final, the stragety and materials resulted from this research would be important for development of edible vaccine expressed in plants stablely or transiently, which might greatly protect young children and animals from the infection of human rotavirus.