蛇毒cystatin在Bac to Bac杆状病毒表达系统的表达与鉴定  

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

作　　者：张晓艳[1] 林旭[2] 林建银[2] 

机构地区：[1]福建医科大学病原生物学系,福州350004 [2]福建医科大学分子医学研究中心,福州350004

出　　处：《福建医科大学学报》2006年第5期419-423,共5页Journal of Fujian Medical University

基　　金：国家自然科学基金资助项目(30371747);福建省科技厅重大项目资助项目(2002Y003)

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Cell-fectin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。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.

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 

分 类 号：Q78[生物学—分子生物学]