致倦库蚊防御素基因的克隆与原核表达及蛋白纯化  

Cloning,Expression and Purification of Defensin Gene from Culex quinquefasciatus

作　　者：王赟[1] 王吉平[2] 张春林[2] 翟素珍[2] 

机构地区：[1]贵阳医学院医学生物技术教研室,贵州贵阳550004 [2]贵阳医学院生物学教研室,贵州贵阳550004

出　　处：《动物医学进展》2012年第11期45-50,共6页Progress In Veterinary Medicine

基　　金：贵州省科学技术基金项目(黔科合J字(2006)2077号);贵州省人才领导小组基金项目(Q2007-2)

摘　　要：以冈比亚按蚊防御素(Defensin)氨基酸序列为种子序列,在致倦库蚊EST库中查找相似序列。通过RT-PCR技术,克隆获得致倦库蚊防御素编码序列。将致倦库蚊防御素成熟肽基因片段定向克隆至原核表达载体pET32a(+),构建致倦库蚊防御素重组表达质粒pET32a-DEF,导入大肠埃希菌Rosetta中表达,并比较不同IPTG浓度、不同诱导时间、不同诱导温度对重组基因表达的影响,以确定最佳诱导表达条件,重组蛋白经His-镍蛋白纯化柱纯化。结果表明,致倦库蚊防御素基因编码区全长300bp,编码99个氨基酸。pET32a-DEF最佳诱导表达条件为,IPTG浓度0.2mmol/L,诱导时间为4h,诱导温度为37℃,经纯化获得大小约29ku的重组蛋白,这为进一步研究该蛋白功能奠定了基础。The defensin of Anopheles gambiae was used as a probe to search for similar genes in EST library of Culex quinquefasciatus.The defensin cDNA of Culex quinquefasciatus was cloned by RT-PCR methods.Mature peptide was cloned into the vector pET32a（＋） to construct recombinant plasmid defensin（pET32a-DEF）.The recombinant plasmid then expressed in E.coli Rosetta.The expression condition was optimized by investigating the effects of concentration of IPTG,induction time and induction temperature.The recombinant product was purified according to the protocol of His-Ni protein miniprep pack.The result showed that the length of defensin gene was 300 bp,encoding 99 amino acid.The optimum induction conditions of pET32a-DEF were as follows：IPTG concentration of 0.2 mmol/L,induction time of 4 h and induction temperature of 37 ℃.The purified fusion protein was about 29 ku,these results laid foundation for further studies on the function of this protein.

关 键 词：致倦库蚊 防御素基因 克隆 原核表达 纯化 

分 类 号：S852.4[农业科学—基础兽医学] R384.1[农业科学—兽医学]