微小隐孢子虫类钙调蛋白基因的克隆与原核表达  

Cloning and Prokaryotic Expression of C.parvum Calmodulin-like Protein Gene

作　　者：杨晓娇[1] 周鹏[1] 米荣升[1] 黄燕[1] 石凯[1,2] 王晓娟[1,3] 王向佩[1,3] 刘宇轩[1] 雷晓思 陈兆国[1] 

机构地区：[1]中国农业科学院上海兽医研究所,农业部动物寄生虫学重点实验室,农业部动物产品质量安全生物性危害因子风险评估实验室(上海),上海200241 [2]四川农业大学动物医学院,四川雅安625014 [3]吉林农业大学动物科学技术学院,吉林长春130118

出　　处：《动物医学进展》2015年第1期26-30,共5页Progress In Veterinary Medicine

基　　金：国家科技重大专项项目(2012ZX10004220);中央级公益性科研院所基本科研业务费专项资金项目(2013JB13);家畜疫病病原生物学国家重点实验室开放基金课题(SKLVEB2013KFKT017);上海市科技兴农重点攻关项目[沪农科攻字(2005)第3-4号]

摘　　要：为了对微小隐孢子虫(C.parvum)类钙调蛋白(CML)基因进行原核表达,分析重组表达蛋白的反应原性。以C.parvum卵囊cDNA为模板,用PCR方法扩增得到C.parvum CML基因。将CML基因连接到克隆载体pMD18-T,获得重组质粒pMD-CML,经限制性内切酶BamHⅠ和XhoⅠ双酶切后,连接到经相同内切酶酶切的表达载体pGEX-6p-1上,构建重组表达质粒,转化到大肠埃希菌BL21(DE3)中进行诱导表达。利用GST亲和树脂法纯化重组表达蛋白,对纯化的重组蛋白进行Western blot分析。结果表明,成功构建了重组原核表达质粒pGEX-CML,重组质粒转化菌经IPTG诱导后成功地表达出了分子质量约为51ku的重组蛋白rCML,纯化的蛋白rCML能与感染兔隐孢子虫(C.cuniculus)的兔血清发生特异性反应,具有很好的反应原性。In order to express Cryptosporidium parvum calmodulin-like protein （CML） gene in E.coli BL21（DE3） and analyze the antigenicity of the recombinant protein,CML gene was amplified by PCR with cDNA of C.parvum oocysts.The amplified CML gene was cloned into pMD18-T vector and the DNA of recombinant pMD-CML plasmid was extracted.The plasmid was digested with double enzymes and the ob-jective fragments were connected with pGEX-6p-1 which had been digested with same enzymes.After iden-tifying by double restrict enzyme digestion and gene sequence analysis, the recombinant plasmids were transformed to E.coli BL21（DE3） cells and the transformed bacteria was induced to express with IPTG. Recombinant proteins were purified by High-Affinity GST·Bind Resin affinity chromatography.Antigen-icity of the recombinant proteins was analyzed by Western blot.The results showed that the prokaryotic expression vector pGEX-CML was constructed successfully and an approximate 51 ku recombinant protein rCML was expressed successfully after inducing with IPTG.The purified recombinant protein could be recognized specifically by the sera from rabbit infected with C.cuniculus.

关 键 词：微小隐孢子虫 类钙调蛋白基因 克隆 原核表达 

分 类 号：S852.723[农业科学—基础兽医学] Q785[农业科学—兽医学]