间日疟原虫醛缩酶的克隆、表达与初步鉴定  

Cloning,expression and identification of Plasmodium vivax aldolase

作　　者：高迎[1,2,3] 陶志勇[1,2] 夏惠[1,2] 陈勇[1,2] 王雪梅[1,2] 方强[1,2] 

机构地区：[1]蚌埠医学院病原生物学教研室,安徽蚌埠233030 [2]蚌埠医学院安徽省感染与免疫重点实验室,安徽蚌埠233030 [3]河南省南阳市中心医院检验科,473000

出　　处：《蚌埠医学院学报》2013年第7期777-780,共4页Journal of Bengbu Medical College

基　　金：卫生部寄生虫病预防与控制技术重点实验室开放课题(WK009-001);安徽省教育厅自然科学研究重点资助项目(KJ2012-A-200)

摘　　要：目的:克隆间日疟原虫醛缩酶(Plasmodium vivax aldolase,PvALD)基因,体外表达和鉴定重组PvALD蛋白。方法:PCR法从间日疟患者血液DNA样品中扩增PvALD基因,构建pET32c-PvALD重组表达质粒,转化大肠埃希菌(E.coli)BL21(DE3+),诱导表达带有His标签的重组蛋白,并对PvALD在E.coli中表达进行优化,观察诱导时间、诱导剂浓度、培养基种类对该蛋白原核表达的影响。采用镍柱亲和层析纯化重组蛋白,相应表达物分别采用SDS-PAGE电泳和Western blot进行分析鉴定。结果:PvALD的PCR产物分子量为1.1 kb,重组质粒pET32c-PvALD经测序验证,其插入片段序列与GenBank参考序列相似性为100%,转化E.coli所表达的重组蛋白分子量约为60 kD。PvALD在LB中1 mmol/L IPTG诱导6 h条件下获得最佳的表达效果。亲和层析纯化后的PvALD经SDS-PAGE电泳检测,呈现单一条带,Western blot分析结果显示,该重组蛋白只能被间日疟患者血清特异性识别,而不能被恶性疟患者血清识别。结论:成功克隆了PvALD基因,表达了重组PvALD蛋白,为以PvALD作为靶标的间日疟快速诊断方法提供了基础。Objective：To clone Plasmodium vivax aldolase （PvALD） gene, express and identify the recombinant PvALD protein in vitro, nethods：PCR was performed to amplify PvALD gene from blood sample of P. vivax infected patient, pET32c-PvALD recombinant plasmid was constructed and transformed into E. coli host BI21 （ DE3 ＋ ）, IPTG was used to induce the recombinant protein PvALD fused with His tag. Induction time, IPTG concentration and media types were optimized for expression of PvALD in E. coll. Then recombinant protein was purified by His-NTA affinity chromatography and identified by SDS-PAGE and Western blot. Results ： The PvALD PCR product was about 1.1 kb, meeting the expectation of predicted fragment size. The recombinant pET32c-PvALD plasmid was verified by sequencing that the insertion was 100% homology with GenBank reference sequence. Optimized condition for recombinant PvALD was at 1 mmol/L IPTG concentration,6 h and in LB medium. SDS-PAGE result showed that recombinant PvALD was about 60 kD. Western blot result showed that recombinant PvALD could be recognized by serum from vivax-malaria patient, but not by serum from falciparum-malaria patient. Conclusions ： The PvALD gene is successfully cloned, and recombinant PvALD protein is expressed,which offers basis for diagnosis rapidly.

关 键 词：间日疟原虫 醛缩酶 原核表达 纯化 亲和层析 

分 类 号：R382.31[医药卫生—医学寄生虫学]