Studies Of Cloning And Prokaryotic Expression On Follicle- Stimulating Hormone β Gene In Moschus Moschiferus

Follicle-stimulating hormone was composed of alpha and beta subunits, and the beta subunit who play an important role in follicle-stimulating hormone biological activity and immunological activity. Studies had shown that FSH beta gene was one of the major genes which affect the animals breeding and musk secretion of the Siberian musk deer(Moschus moschiferus). In this study, as the object to musk deer, cloning, prokaryotic expression used in to FSHβ gene expression in E. coli, preparation of polyclonal antibody. The main results were shown as follows:Selected primers from reference in this study, amplified the complete sequence of FSHβ gene in the musk deer, length 4170bp, coding sequence (CDS) length of 390 bp. Bioinformatics analysis showed that:the FSHβ protein belongs to the glycoprotein gene family and hydrophilic proteins. The alignment results form the CDS sequences of 27 species show that:the identity percent between the Siberian musk deer and the forest musk deer (Moschus berezovskii) is 98.7%; it is 93.2% with sika deer, and 95.5%～97.0% with bovine. Phylogenetic analysis based on FSHβ coding region and amino acid sequence implied that the Siberian musk deer had closer relationship with the forest musk deer.The FSHβ CDS sequences without signal peptide sequence were biosynthesized. The prokaryotic expression vector, called PGEX-6P-1-FSHβ, successfully constructed. It was induced expression with IPTG, and bacterial bodys were broken by ultrasonic wave. SDS-PAGE gel electrophoresis showed that fusion protein about 38KDa, which the product expressed mainly in the form of inclusion bodies. Western blot analysis proved that the recombinant fusion protein had good immunogenicity.Four mices were immunized with purified recombinant protein, and the serum was separated to get the polyclonal antibody. Indirect Elisa test results showed that the titer of antibody is 1:51200.