Expression And Immunological Effect Of Rotavirus VP7 Gene In Arabidopsis Thaliana Seeds

Rotavirus is the primary cause of severe gastroenteritis in children and pups,with 600,000-800,000 estimated annual deaths worldwide in the developing countries.At present,effective treatment of humans or animals infected with rotavirus is still not available.Glucose can temporarily supplement the decrease of body fluid level caused by diarrhea,but it can not fundamentally eliminate viral harm.On this account,the social impact and the economic burden of the family are enormous.Therefore,a safe and effective vaccine against rotaviral infection is urgently required.VP7 gene sequence of the human rotavirus(HRVVP7)was obtained from patent(US 7,285,280B1).HRVVP7 gene sequence was designed and synthesized based on E.coli codon preference,and prokaryotic expression vector pET-22b-HRVVP7 was constructed and successfully expressed in E.coli BL21DE3.The vector was transferred into BL21(DE3)cells of E.coli and engineered stain was finally obtained.HRVVP7 protein had different degrees of expression in the inclusion body and the soluble part.The optimal conditions for the expression of soluble protein were determined as follows: induction temperature 30 ?,induction time 9 h and concentration of IPTG 1.0 mM.The purified HRVVP7 protein was obtained by affinity chromatography with Ni2+.Based on Arabidopsis codon preference,HRVVP7-linker-CTB gene was designed and synthesized.Using it as template,the optimized HRVVP7 nucleic acid sequence was obtained by PCR method,and plant expression vectors pPhaP3301-HRVVP7-linker-CTB and pPhaP3301-HRVVP7 were successfully constructed.With Agrobacterium tumefaciens mediated infection of Arabidopsis thaliana,positive transgenic Arabidopsis thaliana plants were obtained by glyphosate screening.High expression T3 generation seeds of transgenic Arabidopsis thaliana were detected and quantified by Western blotting and ELISATo explore multiple ways of HRVVP7 production in plant bioreactor,the technique of tissue culture of safflower was used to transfer pPhaP3301-HRVVP7 into cotyledons of safflower by Agrobacterium tumefaciens.The surviving plants were verified by PCR,and the positive plants continued to propagate after harvesting seeds.The transgenic safflower seeds of T3 generation with stable inheritance were harvested,HRVVP7 protein was detected and qualitatively determined by SDS-PAGEG and Western blotting,and the quantitative analysis of ELISA was used to provide a new feasible way of plant production of rotavirus vaccine.The soluble total protein was extracted from transgenic Arabidopsis thaliana seeds containing HRVVP7-linker-CTB and HRVVP7.Mice were immunized after content determination,and the immunological effect of the plant vaccine prepared in the experiment was analyzed.In HVVP7-linker-CTB and HRVVP7 transgenic Arabidopsis thaliana seed protein samples,the immunized mice could produce saliva,feces,intestinal mucosal IgA antibody and serum IgG antibody;Serum IgG antibody had good virus neutralization ability;Oral delivery of plant-expressed HRVVP7-linker-CTB and HRVVP7 to female mice provided passive protection from rotaviral challenge to their suckling neonatal progeny.The above results show that the fusion of HRVVP7 with CTB protein has better immunological effect than HRVVP7 protein alone in the animal immunization experiment.In this study,HRVVP7(US 7285280B1)gene was first expressed in Arabidopsis thaliana and safflower,and the immunological effects before and after CTB fusion were compared.The study will lay a foundation for the production of rotavirus subunit vaccinein in plant bioreactor.