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目的:通过定点突变,构建集成干扰素(IIFN/165S),以期获得高效的新型药物分子。方法:采用PCR体外定点突变技术,使集成干扰素IIFN基因的第165位密码子由CGT突变为AGT。扩增片段克隆入pET-23b表达载体,重组质粒转化大肠杆菌BL21(DE3)。在LB培养基中培养,经IPTG诱导表达的IIFN/165S经包涵体变性、复性以及层析纯化后,经SDS-PAGE、Western blot和MALDI-TOF-MS分析,用WISH-VSV系统进行抗病毒活性测定同时应用流式细胞术检测细胞凋亡率。结果:IIFN/165S以包涵体形式表达。纯化后,IIFN/165S的纯度大于95%,分子量为18172,比活性(7.63±0.22)×108IU/mg,诱导细胞凋亡率呈剂量依赖。结论:构建了IIFN/165S的表达载体,并成功地在大肠杆菌中表达,获得了高纯度高活性突变分子IIFN/165S。
OBJECTIVE: To construct an integrated interferon (IIFN / 165S) by site-directed mutagenesis in order to obtain highly efficient new drug molecules. Methods: The site - directed mutagenesis of PCR was used to mutate the codon 165 of the integrated interferon IIFN gene from CGT to AGT. The amplified fragment was cloned into pET-23b expression vector and the recombinant plasmid was transformed into E. coli BL21 (DE3). IIFN / 165S induced by IPTG was denatured, renatured and purifed by IPTG, then analyzed by SDS-PAGE, Western blot and MALDI-TOF-MS and analyzed by WISH-VSV system The virus activity was determined by flow cytometry. Results: IIFN / 165S was expressed as inclusion bodies. After purification, the purity of IIFN / 165S was more than 95%, the molecular weight was 18172, and the specific activity (7.63 ± 0.22) × 108IU / mg, which induced the apoptosis rate in a dose-dependent manner. Conclusion: The expression vector of IIFN / 165S was constructed and successfully expressed in Escherichia coli. The high purity and high activity mutant IIFN / 165S was obtained.