西司他汀类;_克隆细胞;_Sf9细胞;_杆状病毒科;

蛇毒cystatin在Bac to Bac杆状病毒表达系统的表达与鉴定

http://www.100md.com 《福建医科大学学报》 2006年第5期

西司他汀类;,克隆细胞;,Sf9细胞;,杆状病毒科;,基因,病毒;,基因表达;,蛇毒液类,,西司他汀类;,克隆细胞;,Sf9细胞;,杆状病毒科;,基因,病毒;,基因表达;,蛇毒液类,蛇毒cyst

     摘要: 目的探讨蛇毒cystatin在Bac to Bac杆状病毒表达系统中的表达。方法 PCR扩增蛇毒cystatin基因,将其克隆到pFastBacHTc中,通过转化E.coliDH10Bac筛选克隆,抽提重组Bacmid/cystatin,后者经Cellfectin介导转染Sf9细胞,获取重组病毒,扩增病毒并感染Sf9细胞进行表达,SDSPAGE、Westernblot分析鉴定表达蛋白66。结果获得重组cystatin的杆状病毒,Sf9细胞能表达出与蛇毒cystatin单抗、5×His单抗结合的蛋白,相对分子质量约15 kD。结论蛇毒cystatin在Bac to Bac杆状病毒表达系统中成功表达。

    关键词: 西司他汀类; 克隆细胞; Sf9细胞; 杆状病毒科; 基因,病毒; 基因表达; 蛇毒液类

    Expression and Identification of Cystatin from Snake Venom in BactoBac Baculovirus Expression System

    Zhang Xiaoyan, Lin Xu, Lin Jianyin

    1.Division of Etiological Biology,2.Research Center of Molecular Medicine, Fujian Medical University, Fuzhou 350004, China

    ABSTRACT: Objective To investigate the expression of cystatin from snake venom in BactoBac baculovirus expression system. Methods The cystatin gene was amplified by PCR. The recombinant plasmid was constructed by inserting cystatin gene sequence into the plasmid pFastBacHTc. The plasmid pFast/cystatin was transformed into DH10Bac E.coli to construct and obtain the recombinant vector Bacmid/cystatin. Bacmid/cystatin transfected into Sf9 cells via cellfectin to generate the recombinant baculovirous. The expressed protein was analyzed and identified by SDSPAGE and Westernblot. Results The recombinant baculovirous was obtained. The recombinant cystatin protein was expressed in Sf9 cells and detected by Westernblot using monoclonal antibody of cystatin or 5 His monoantibody. The molecular weight of the recombinant cystatin protein was about 15 000 Dalton. Conclusion The recombinant cystatin protein was successfully expressed in BactoBac baculovirus expression system.KEY WORDS: cystatins; clone cells; Sf9 cells; baculoviridae; genes,viral; gene expression; snake venoms ......

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