Expression Of Human Rotavirus VP7 Protein In Eukaryotic And It's Fermentation Condition Optimization

Rotavirus is a double-stranded ribovirus that belongs to the genus Reovirus.At present,studies have shown that the single major cause of diarrhea in infants and young children is caused by rotavirus infection.After the infection,the body produces enterotoxin,which can cause serious children.Diarrhea can even cause death due to dehydration.The VP7 structural protein encoded by the structural gene vp7 is a glycoprotein on the outer surface of rotaviruses.It can induce the production of IgG antibodies and IgA antibodies and is associated with the protection of immunity.It serves as the basis for judging the serotype of rotavirus G.Although some live attenuated rotavirus vaccines can increase the infant's resistance after vaccination,there are currently no specific drugs that can treat rotavirus infections,and because there are different types of rotaviruses in different regions.Different virus epidemic strains or epidemic strain combinations,which all have different immunogenicity,are of great significance for the development of safe and effective protein vaccines against different types of rotaviruses.The purpose of this experiment is to prepare for the development of rotavirus protein vaccines and provide technical support for the suppression of rotavirus transmission in China.Because the virus cannot be cultured in vitro,the vp7 gene in the serotypes G1,G2,and G3 of the rotavirus strain was transformed into Pichia pastoris GS115 expression system.In Pichia pastoris GS115,transformants that have bee n transformed by initially screened,and then induce the expression of the target protein VP7 protein using methanol,followed by detection at the DN A,RNA,and protein levels,orthogonal experiments were carried out to optimize the fermentation conditions for the detection of successful transmutants.Primers were designed and the three plasmids provided by the company were used as templates to clone the target fragments of three vp7 genes,and the target fragment gel recovery products were respectively connected with the pMD18-T vector.The pMD18-T-G1,pMD18-T-G2,and pMD18-T-G3 were double digested with Not I and EcoR I,and were then ligated with the same double-digested plasmid pPIC9 K to successfully construct three recombinant expression vectors.The three recombinant plasmids were transformed into Pichia pastoris by electroporation,and then screened by MD medium.After PCR amplification,real-time quantitative PCR and Western Blotting were used for further detection,only 3 Mut⁺positive G2 types were screened out.Transformants,G1 and G3 transformants were not successfully screened.Finally,by using the method of orthogonal experiment of pichia induced to optimize the conditions of target protein,first carries on the inductio n time,medium initial pH,medium methanol concentration,induction temperature of four single factor experiment,the optimal conditions were selected in each factor three a nd then make orthogonal experiment of L₉?3⁴?,set up three levels in all types of experiments.According to the analysis of the results,the influencing factors affecting the expression level of the target protein were induction temperature>induction time>medium methanol concentration>medium initial p H,and the optima l induction condition was28°C,the initial pH of the medium was 4.0,the concentration of methanol in the medium was 3%,and the expression of the target protein was highes t at the induction time of 72 h.And the concentration was 0.142 mg/ml.