集成干扰素突变体IIFN/165S的构建及其生物学评价

中国生物工程杂志

2013, Vol. 33

Issue (7): 8-12

研究报告

集成干扰素突变体IIFN/165S的构建及其生物学评价

田硕¹, 姚文兵², 徐晨³

1. 中国医学科学院基础医学研究所北京协和医学院基础学院医学分子生物学国家重点实验室北京 100005;
2. 中国药科大学 210009;
3. 北京三元基因工程有限公司北京 102600

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

TIAN Shuo¹, YAO Wen-bin², XU Chen³

1. Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, National Laboratory of Medical Molecular Biology, Beijing 100005, China;
2. School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, China;
3. Beijing Tri-prime Genetic Engineering Co.Ltd., Beijing 102600, China

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摘要： 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)×10⁸ IU/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。

关键词： 集成干扰素; 定点突变; 高效; 抗肿瘤

Abstract: 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.

Key words: Integrated interferon Site-directed mutagenesis High potency Anti-tumor

收稿日期: 2012-12-11 出版日期: 2013-07-25

ZTFLH:

Q78

基金资助: 北京市科委基础研究专项课题资助项目(Z0005187040621)

通讯作者: 徐晨 E-mail: xuchen@triprime.com

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引用本文:

田硕, 姚文兵, 徐晨. 集成干扰素突变体IIFN/165S的构建及其生物学评价[J]. 中国生物工程杂志, 2013, 33(7): 8-12.

TIAN Shuo, YAO Wen-bin, XU Chen. Construction and Biological Assay of Integrated Interferon Mutant IIFN/165S. China Biotechnology, 2013, 33(7): 8-12.

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I7/8

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