重组细胞色素酶P450 3A4表达和鉴定

Expressed and Purified Recombinant Human Cytochrome P450 3A4

细胞色素酶P450是代谢内源性物质和外源性物质的重要的酶,在药物治疗和药物开发领域以及了解潜在的毒性物质和致癌性物质的代谢机制起决定性作用。旨在构建细胞色素酶P450 3A4的表达载体,在大肠杆菌中表达并纯化得到CYP3A4蛋白。用逆转录-聚合酶链反应方法从人肝脏总RNA中得到CYP3A4的cDNA,然后直接插入pMD20-T Vector中,将测序正确的N-末端和C-末端进行修饰。然后利用双酶切插入到表达载体pET-28a-c(+)Vectors,转化到大肠杆菌BL21(DE3)中表达。并通过定点突变的方法获得CYP3A4亚型P467S(CYP3A4*19)。采用SPSS13.0设计4因素2水平正交实验,对α-ALA(0.5 mmol/L和1 mmol/L)、IPTG(0.5 mmol/L和1 mmol/L)、卡那霉素(50μg/mL和100μg/mL)添加浓度及菌的接种密度(接种1%和接种2%)进行优化。并用SPSS13.0对结果进行分析,选择出比较好的组合进行下一步的大规模的诱导表达。经定点突变的方法成功获得了CYP3A4*19亚型。经IPTG诱导获得CYP3A4蛋白,并通过Western blot验证。用BCA蛋白浓度定量分析试剂盒测定的膜蛋白浓度在65μg/mL左右,α-ALA、抗生素(卡那霉素)、T7启动子诱导剂IPTG及接种密度高低两水平中对膜蛋白的表达水平的影响没有统计学意义。

Cytochrome 17450 is an important enzyme for metabolism of endogenous substances and exogenous substances, and plays a decisive role in drug treatment, drug development and understanding the metabolism of potential toxic substances and carcinogenic substances. In order to construct the expression vector of cytochrome P450 3A4 ,expressed and purified CYP3A4 protein in Escherichia coli ,reverse transcription-polymerase chain reaction was used to obtain CYP3A4 cDNA from human liver total RNA, and then inserted directly into the pMD （~ 20-T Vector. The correct sequencing was modified with N-terminal and C-terminal that have been conducive to the expression. After double digestion the CYP3A4 gene was inserted into the expression vector pET-28a-c （ ＋ ） vectors and transformed into E. coli BI21 （DE3） to express. CYP3A4 mutation subtype of CYP3A4 ＊ 19 was obtained by site-directed mutagenesis. Four factors and two levels of orthogonal experiment designed by SPSS13.0 to optimize four factors of α-ALA （0.5 mmol/L and 1 retool/L） ,IPTG （0. 5 mmol/L and 1 mmol/L） ,kanamycin （50 μg/mL and 100 μg/mL） concentration and bacteria inoculation density （inoculation 1% and inoculated with 2% ） for portent expression. The results were analyzed using SPSS13.0 to select a good combination of large-scale induced expression. CYP3A4 protein was induced by IPTG, and verified by Western blot. Membrane protein concentration is around 65 μg/mL. The level of α-ALA, antibiotics （kanamycin）, IPTG,inoculation density on the level of expression of membrane proteins was not statistically significant. Expression of membrane proteins was verified by Western blot for recombinant CYP3A4 protein. The cloning of cytochrome P450 3A4 protein was obtained that laid the foundation for drug interaction experiments in vitro.