摘要
目的 构建泡球蚴18(Em18)基因的原核表达质粒,获得高效表达、有生物活性的Em18重组蛋白,为包虫病诊断试剂的研制奠定基础。 方法 用DNAman软件设计引物,分别在引物5′端和3′端添加EcoRⅠ和 XhoⅠ酶切位点,以pMD18 T/Em18原核表达质粒为模板,PCR扩增Em18基因片断,经酶切,克隆入原核表达载体 pET 41a(+),构建原核表达质粒 pET41a Em18;转化大肠埃希菌BL21(DE3),酶切、PCR及测序鉴定其插入序列的正确性。经 IPTG诱导表达 rEm18 GST重组蛋白和GST重组蛋白,用谷光甘肽 Sepharose 4B亲合层析柱分别进行纯化,通过 SDS PAGE和Western blot试验分析鉴定。 结果 测序表明构建的 pET41a Em18 原核表达质粒均为正确连接,插入 Em18 基因片断为486 bp。SDS PAGE检测表明Em18基因以 rEm18 GST重组蛋白的方式得到成功表达,在相对分子质量单位 50ku处有表达条带;Western blot分析显示,rEm18 GST重组蛋白能被泡型棘球蚴病人阳性血清识别,具有良好的抗原性。 结论 成功构建了 pET41a Em18原核表达质粒,获得的 rEm18 GST重组蛋白具有生物活性和抗原性,有望应用于泡型包虫病诊断试剂的研制。
Objective To construct the prokaryotic expression plasmid pET41a Em18 and to express and purify Em18 GST fusion protein, so as to lay a foundation for diagnostic kit of echinococcosis. Methods Using recombinant plasmid pMD18 T/Em18 as the template, the Em18 cDNA with EcoRⅠ and XhoⅠ restriction digestion sites was amplified by PCR and subsequently inserted into the prokaryotic vector, pET 41a(+). The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and was then identified by PCR, double enzyme digesting and sequencing. The rEm18 GST fusion protein and recombinant GST protein were expressed by induction with IPTG. The expressed recombinant proteins were purified using a Glutathione Sepharose 4B column. Purification of the target protein was confirmed by SDS PAGE and Western blotting. Results The sequencing analysis indicated that the Em18 was successfully inserted into the expression vector pET41 and the reading frame of the pET41a Em18 was correct and the insert Em18 DNA length was 486 bp. SDS PAGE showed that the rEm18 GST recombinant protein has been expressed successfully and the molecular weight was about 50 ku. Western blot result confirmed that the recombinant protein could specifically react with the serum samples from patients with alveolar echinococcosis (AE) and had good antigenicity. Conclusion pET41a Em18 was successfully constructed and the rEm18 GST fusion protein was obtained. Recombinant Em18 antigen has potential for use in the research of diagnosis for alveolar echinococcosis and the possible development of a diagnosis kit.
出处
《中国寄生虫病防治杂志》
CSCD
2005年第1期33-36,共4页
Chinese Journal of Parasitic Disease Control
基金
国家自然科学基金项目(No.30360097
No.30160081)
新疆重点实验室开放课题基金项目(XJDX0202 2003 01)。
关键词
泡球蚴
Em18基因
原核表达
鉴定
Echinococcus multilocularis
Em18 antigen cDNA
prokaryotic expression
recombinant protein
