摘要
根据GenBank上登录的绵羊抑制素α亚基基因序列,设计合成2对引物,以新疆细毛羊卵巢提取的总RNA为模板,采用RT-PCR方法扩增获得了约812 bp片段,经pMD18-T载体克隆和测序分析证实为新疆细毛羊抑制素α亚基前体基因。进一步通过PCR反应扩增新疆细毛羊INHα亚基基因片段,经过NcoⅠ和HindⅢ双酶切后定向克隆于pET32a原核表达载体,获得pET-INH重组表达载体。将携带有重组表达载体的大肠杆菌BL21(DE3)通过1 mmol.L-1IPTG进行诱导表达,经过SDS-PAGE电泳检测,显示诱导表达蛋白大约为32 ku,与预期表达蛋白大小一致。Western blot检测表明重组原核表达载体在大肠杆菌中成功表达出了目的融合蛋白。新疆细毛羊抑制素α亚基基因的克隆、表达为进一步研究其功能和应用奠定了基础。
The inhibin α subunit was amplified from ovary of Xinjiang Fine-wool sheep by RT-PCR using two pairs of primers which were designed and synthesized according to the sheep inhibin α subunit gene sequence in GenBank. The gene about 812 bp by RT-PCR was inserted into pMD18-T vector and sequencing, the result of sequence analysis show.ed that the gene is Xinjiang Fine-wool sheep inhibin a subunit precursor. The gene of mature inhibin a subunit was cloned by PCR, which was double-digested by Nco Ⅰ and Hind Ⅲ and directionally cloned into the pET32a vector. The recombinant expression plasmid in the E. coli BL21 （DE3） was induced with 1 mmol · L^-1 IPTG. SDS-PAGE analysis showed that the induced expressed protein was about 32 ku. Western blot revealed that the expressed protein was the target fusion protein, which indicated that the recombinant prokaryotic expression vector expressed the fusion protein successfully. The cloning and expression of Xinjiang Fine-wool sheep inhibin α subunit made a foundation for the study of function and application.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2009年第1期15-19,共5页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
关键词
新疆细毛羊
抑制素α亚基
基因克隆
表达
Xinjiang Fine-wool sheep
INH subunit
gene cloning
expression
