猪抑制素α亚基基因克隆及其原核表达

Cloning and prokaryotic expression of Lantang porcine inhibin α-subunit gene

为了克隆猪抑制素α亚基基因和表达其重组蛋白质,本试验采集发情期蓝塘猪卵巢组织,并从中快速抽提总RNA,以此作为模板并根据猪抑制素α亚基基因编码区序列(GenBank号:NM214189)设计合成1对引物,经反转录扩增获得了猪抑制素α亚基基因编码区全长序列。另外,再设计1对特异性引物以扩增抑制素α亚基成熟肽cDNA,并将扩增产物克隆到表达载体pRSET A的BglⅡ和EcoR I酶切位点之间,构建重组质粒pINH-SCAU并转化Escherichia.coli BL21(DE3)株。转化重组质粒pINH-SCAU的重组菌经IPTG诱导后表达的重组蛋白质分子量为20 000,经Ni-NTA凝胶纯化和兔抗牛抑制素α亚基抗体的特异性免疫反应,证明为抑制素α亚基重组融合蛋白质。对蛋白质表达条件的比较,结果显示重组菌经常规诱导剂0.05 mmol/L IPTG诱导5 h后的细菌生长密度OD600值为2,抑制素α亚基重组融合蛋白质的最高表达量为总菌体蛋白质量的32.00%。采用自动诱导培养基替代IPTG,在诱导18 h后能使菌液的OD600达13,同时自动诱导的抑制素融合蛋白质表达量占菌体总蛋白质的32.57%。因此利用自动诱导培养基可以获得较佳的抑制素重组整合蛋白质。

To clone the porcine inhibin α-subunit gene and express its recombinant protein, total RNA was extracted from Lantang porcine ovary tissue by Trizol, and a pair of primers was designed according to the published porcine inhibin α-subunit gene sequence （GenBank No. NM214189）. Inhibin α-subunit gene coding sequence was amplified by RT- PCR. The mature peptide cDNA was further amplified and cloned into the Bgl II and EcoR Ⅰsites of the pRSET A expressing vector to generate the recombinant plasmid pINH-SCAU. The plasmid was transformed into Escherichia. coli BL21 （DE3）, and the transformed bacterium was induced with IPTG to express a recombinant protein with a molecular mass of 20 000, which was verified to be recombinant inhibin subunit fusion protein by 50% Ni-NTA agrose chromatography purification, and by rabbit anti-bovine inhibin α subunit antibody in Western blotting. The highest expression level of 32.00% to the total bacterial protein was achieved following the induction by 0.05 mmol/L IPTG for 5 h, with bacteria growth level at 2 of OD600 value. The expression level of 32.57% was reached, after auto-induction for 18 h, with a bacteria growth level at 13 of OD600 value. Therefore, auto-induc-tion fermentation method is more favorable for the expres- sion of recombinant inhibin α-subunit fusion protein.