猪卵泡抑素基因成熟肽序列的克隆及其在大肠杆菌中的表达

根据猪卵泡抑素(follistatin,FS)基因编码区序列(GenBank登录号:M36512.1)设计并合成1对引物,经PCR扩增获得了FS基因成熟肽序列,将其克隆入pMD18-T载体并转化E.coli DH5α感受态细胞,进行PCR、双酶切及测序验证。然后将其克隆到表达载体pRSET-A的BamHⅠ和HindⅢ酶切位点之间,构建重组质粒pR-FS并转化E.coli BL21(DE3) 感受态细胞,再经PCR、双酶切和测序验证。转化重组质粒pR-FS的重组菌经IPTG诱导后的表达产物进行SDS-PAGE分析,分子质量与预期相符,为26.1 ku左右,经0.2 mmol/L IPTG诱导3 h之后表达量达到最高,约占总菌体蛋白的30%。表达产物可经Ni-NTA凝胶纯化。以上结果表明正确完成了猪FS基因成熟肽序列的克隆及其在大肠杆菌中的表达及纯化。

Cloning of Porcine Follistatin Gene Mature Peptide Sequence and Expression in E.coli BL21(DE3)

A pair of primers was designed according to the porcine follistatin(FS) gene mature peptide sequence(GenBank:M36512.1), the mature peptide sequence of FS gene was achieved by PCR. The PCR product of the expected length was cloned into the pMD18-T vector and transformed into E.coli DH5α. The positive clones were identified by PCR, enzyme digestion and sequencing. Then insert the FS gene mature peptide between the BamHⅠ and HindⅢ sites of the pRSET-A vector generating the recombinant plasmid pR-FS and transformed it into E.coli BL21(DE3). The positive clones were also identified by PCR, enzyme digestion and sequencing. Bacteria transformed by pR-FS were expressed in E.coli BL21(DE3) after induction with IPTG and produced a product showing expected molecular mass of 26.1 ku in SDS-PAGE, which achieved the highest level induced by 0.2 mmol/L IPTG for 3 h. The product can be purified by 50% Ni-NTA agarose. These results demonstrated that the porcine FS gene mature peptide sequence was successfully cloned and expressed in E.coli BL21(DE3).