The Cloning Of Liangshan Semi-fine Wool Sheep Inhibinα-subunit Gene And The Construction Of Mature Peptide Dimer

Inhibin(INH) is a disulphide-linked heterodimeric glycoprotein consisting of a-and β-subunits. For its inhibition on the synthesis and secretion of follicle-stimulating hormone (FSH), active immunization against INH could increase FSH level, promoted follicle development, increased ovulation rate and lambing percentage of females, increased sperm production and made males come to puberty earlier which have great scientific value on improving animal reproductivity.INH-a subunit was amplified from ovary of Liangshan Semi-fine wool sheep by RT-PCR using a pair of primers which were designed according to Xinjiang fine wool sheep INH-a subunit gene sequence and inserted into the pMD19-T Simple Vector for bioinformatics analysis. The length of cDNA is857bp, however, only402bp encode134amino acids to consist mature peptide. Mature peptide is a hydrophilic extrinsic protein, having the transforming growth factor-β (TGF-β) conserved domain and epitope region (1-32AA). The INH-a subunit is highly conservative between species suggesting its correlation with some important physiological function.Take INH-a subunit mature peptide encoding gene as a unit and insert it between BamH Ⅰ and Sac Ⅰ sites in plasmid pRSET A to generate monomer plasmid pR-INH. According to the isocaudamer enzyme principle, the unit could be inserted between Bgl Ⅱ and Hind Ⅲ sites in recombinant plasmid pR-INH directionally by double digestions BamH Ⅱ Hind Ⅲ and Bgl Ⅲ Hind Ⅲ. Both monomer and dimer protein could be expressed from E. coli BL21(DE3) by induction. The fusion proteins were purified by osmotic shock and their activity ware detected using Western Blot. The results demonstrated that fusion priteinsexpressed by pR-INH and pR-2INH recombinant plasmids have the immunogenicity of INH, and thus form the strong basis for the developement of highly-efficient vaccine in the follow up study.