The Cloning, Sequencing Analysis And Expression Of INHα Subunit Gene Of Xinjiang Fine-wool Sheep

Inhibin is a disulphide-linked heterodimeric glycoprotein consisting ofαandβsubunits,mainly produced by the sex gland of animal,which is a member of the transforming growth factorβ(TGF-β) superfamily.In vivo,inhibin has biological activity in suppressing the synthesis and releasing of FSH.The emphasis of this study is focused on cloning and fusion expression of mature INHα-subunit cDNA of Xinjiang fine-wool sheep in BL21(DE3) strain.The aim of the present study is to provide data for studying the effect of immunization of INH recombinant protein on animal reproduction.The inhibinα-subunit was amplified from ovary of Xinjiang free-wool sheep by RT-PCR using two pairs of primers which were designed and synthesized according to the New Zealand sheep inhibinα-subunit gene sequence published in GenBank (L28815).The gene about 800bp by RT-PCR was inserted into pMD18-T vector and sequencing,the result showed that the gene is Xinjiang fine-wool sheep inhibin a-subunit.The length of cDNA is 812bp,encoding a predicted precursor protein of 265 amino acids.The cDNA and its predicted polypeptide of Xinjiang fine-wool sheep inhibin a-precursor are considerably homologous to New Zealand sheep counterparts(98.6%and 98.1%,respectively),and compared with their cDNA of matureα-subunit,they differed in only one nucleotide at the position 303(T to C),this is a synonymous mutations.The obtainedα-subunit cDNA sequences with corresponding sequences of mammal reported in GenBank was compared with DNAstar software.The results showed that 96.0%,88.1%,87.8%,85.8%,84.2%and 82.0%identity of the matureα-subunit cDNA of Xinjiang fine-wool sheep inhibin to that of cow,pig,horse,human,mice and rat,respectively.The mature inhibinα-subunit consisting of 131 amino acids with 14.7ku molecular weight,includes one potential N-glycosylation site,two potential protein kinase C phosphorylation site, three potential Casein kinaseⅡphosphorylation site and 7 evolutionarily conserved cysteine residues,an antigenic determinant of matureα-subunit region was in 1 to 32 amino acid sequence.The mature inhibinα-subunit cDNA(411bp) of Xinjiang fine-wool sheep was amplified by PCR,using as template plasmid pMD-INH.The expression fragment, producing from PCR product by NcoⅠand HindⅢ,was ligated with the prokaryotic expression expressing vector pET-32a(+)which was also digested by NcoⅠand HindⅢ,and then the ligation was transformed to E.coli DH5α.The result of sequencing of plasmid proved that recombination vector was obtained(named pET-INH).The recombinant expression plasmid in the E.coli BL21(DE3) was induced with IPTG in lower temperature(30℃).SDS-PAGE analysis showed that the induced expressed protein was about 32ku.Western blotting revealed that the expressed protein was the His tag fusion protein,which indicated that the recombinant prokaryotic expression vector expressed the fusion protein successfully.The fusion protein was purified by the process of osmotic shock,and the targeted protein was obtained.The results further indicated that it is feasible to obtain antigen using this method.