细胞色素P450 4F2基因的克隆、突变、表达和纯化

Clone and Expression CYP4F2 Gene and Its Variants

目的构建人细胞色素P450 4F2基因野生型、V81G和V433M突变型表达载体,在大肠杆菌中表达并纯化出CYP4F2蛋白。方法用RT-PCR方法从人肾脏总RNA中逆转录扩增CYP4F2,将其插入克隆载体pMD18-T Simple Vector中,将测序鉴正确的CYP4F2基因N、C端改造,克隆构建重组表达载体pCWori+-CYP4F2(His)_4-WT。同时采用重叠PCR定点突变法构建pCWori +-CYP4F2 V81G(His)_4和pCWori+-CYP4F2 V433M(His)_4表达载体,分别转化至E.Coli XL-Blue中表达。结果经IPTG诱导可有效表达CYP4F2和突变体V81G与V433M,经Western blot分析可以观察到分子量为57 kD的诱导表达条带,该表达蛋白可与His-Probe多克隆抗体起特异反应。用His- bind亲和层析纯化得到了纯度较高的His融合蛋白。结论用基因工程技术在大肠杆菌中有效表达人CYP4F2基因野生型、V81G和V433M突变型蛋白、并得到了纯度较高的CYP4F2蛋白,为进一步研究其生物学功能及研制相应抗体奠定了基础。

Objective To construct the expression vectors of human cytochrome P450 （CYP） 4F2 wild type, V81G and V433M Variants and express and purify CYP4F2 protein in E. Coli XL-Blue. Methods The entire coding region of CYP4F2 gene was cloned from total RNA isolated from human kidney tissue and verified by sequencing. N and C terminal region of DNA fragments were modified and subcloned into pCWori ＋ Vector to facility expression, pCWori ＋-CYP4F2 V81G（His）4 and pCWori ＋-CYP4F2 V433M（ His）4 were constructed using overlap PCR site-directed mutagenesis method and expressed in E. Coli XL-Blue. Results After IPTG induction, CYP4F2 wild type and variants were verified by Western blot analysis. Pure recombinant CYP4F2 protein was obtained by High-affinity His-bind Purification system. Conclusion We cloned CYP4F2 to expression the wide type and variants protein in E. Coli XL-Blue in order to study the biological function and make antibody of CYP4F2.