集成干扰素突变体IIFN/165S的构建及其生物学评价

Construction and Biological Assay of Integrated Interferon Mutant IIFN/165S

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:Construct IIFN/165S through site-directed mutagenesis in order to obtain a new type of molecule with higher potency.Methods: CGT was substituted for AGT at position 165 of integrated interferon through PCR site-directed mutagenesis in vitro.The Amplified fragment was constructed in pET23b expression vector,and transformed into E.coli BL21(DE3) pLysS.The recombinant protein was purified and the purified protein was analyzed by SDS-PAGE,Western blot and MALDI-TOF-MS.The anti-virus was determined by WISH-VSV system.The apoptosis rate was detected by flow cytometry.Result: IIFN/165S was expressed as inclusion bodies with the yield of more than 30% of total bacterial protein.The purity of IIFN/165S was more than 95% with IIFN immunogenicity,and the molecular weights of IIFN/165S was 18172.The biological activity was(7.63±0.22)×108×106 IU/mg.IIFN/165S induced Daudi cells apoptosis in a dose-dependent manner.Conclusion: The construction,expression and purification technology of IIFN/165S had been established.