集成干扰素突变体Ⅱ的分子构建、表达及提纯

中国生物工程杂志

研究报告

集成干扰素突变体Ⅱ的分子构建、表达及提纯

牛晓霞刘金毅杨轶吴晓东

中国科学院研究生院北京三元基因工程有限公司北京三元基因工程有限公司

Construction，expression and purification of Recombinant Consensus Interferon Mutant Ⅱ

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摘要： 目的：通过定点突变，构建集成干扰素突变体Ⅱ（IFN-Con-m2），以期获得兼具高效作用和可定点聚乙二醇（PEG）修饰的新型药物分子。方法：采用PCR体外定点突变技术，使集成干扰素突变体Ⅰ（IFN-Con-m1）基因的第86位密码子由TAC突变为TGC。将扩增片段克隆入pET-23b表达载体，重组质粒转化大肠杆菌BL21（DE3）。IPTG诱导后，表达的IFN-Con-m2经包含体变复性、疏水层析、DEAE层析和凝胶过滤层析等纯化后，用WISH-VSV系统进行生物活性测定。结果：IFN-Con-m2以包涵体形式表达，表达量占菌体总蛋白的30%以上。纯化后，IFN-Con-m2的纯度大于95%，比活性大于5.0×108IU/mg。结论：构建了IFN-Con-m2的表达载体，并成功地在大肠杆菌中表达，获得了高活性突变分子IFN-Con-m2，建立了IFN-Con-m2的纯化工艺。

关键词： 构建; 表达; 纯化; 集成干扰素突变体; 定点突变

Abstract: Interferon alpha are used clinically to treat a variety of viral diseases and cancers. They have a short circulating half-life, which necessitates frequent administration to patients. Previous studies showed that it is possible to extend the circulating half-life of interferon alpha by modifying cysteine residues of the protein with poly(ethylene glycol)(PEG) reagents. But protein seldom have unpaird -SH, so we introduce a free cysteine residue into the protein by recombinant DNA technique. Recombinant consensus interferon mutant Ⅰ（IFN-Con-m1）belongs to the family of interferon alpha. It has a wide variety of biological effects that include antiviral, antiproliferative, and immunomodulatory activities. Recombinant consensus interferon mutant Ⅱ (IFN-Con-m2 ) was a cysteine mutant of IFN-Con-m1. The protein has higher specific activity and may be Site-directed modified by PEG. It was constructed by substitution of Tyr at position 86 with Cys using site-directed mutagenesis. The DNA was constructed in pET-23b expression vector, and transformed into E.coli BL21（DE3）. IFN-Con-m2 was expressed as inclusion bodies with the yield of more than 30% of total bacterial protein. The recombinant protein was expressed after IPTG induction and purified by Phenyl Sepharose, DEAE Sepharose FF and Sephacryl S-100. After purification, the purity of IFN-Con-m2 was higher than 95%, and the biological activity was more than 5.0×108IU/mg. Western blot displayed the recombinant product had strong immunological activity with mouse anti-human IFN-Con-m2 monoclonal antibody.

Key words: Construction Expression Purification Consensus interferon mutant Site-directed mutagenesis

收稿日期: 2006-08-11 出版日期: 2006-12-25

通讯作者: 牛晓霞

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引用本文:

牛晓霞,刘金毅,杨轶,吴晓东. 集成干扰素突变体Ⅱ的分子构建、表达及提纯[J]. 中国生物工程杂志, .

. Construction，expression and purification of Recombinant Consensus Interferon Mutant Ⅱ. China Biotechnology, .

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2006/V26/I12/1

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