Em18基因_____泡球蚴；_Em18基因；

泡球蚴18基因的克隆、原核表达质粒的构建及其初步表达的研究

http://www.100md.com 《新疆医科大学学报》 2005年第1期

Em18基因,,泡球蚴；,Em18基因；,重组蛋白,1材料与方法,2结果,3讨论,参考文献：

     摘要：目的: 克隆、分析泡球蚴18(Em18)抗原基因，构建pET41aEm18原核表达质粒，并初步诱导表达Em18重组蛋白。方法: DNAman软件设计引物，RTPCR法克隆Em18 cDNA并构建pMD18T/Em18质粒，测序确定序列，利用DNAman和BLAST软件，对其进行基因序列分析。构建pET41aEm18原核表达质粒，测序鉴定插入序列正确性。IPTG初步诱导和表达rEm18GST重组蛋白，SDSPAGE电泳检测。结果: 测序结果显示Em18抗原基因长度为486 bp，编码161个氨基酸。BLAST比对分析表明为一新序列并被GenBank收录(AY513691)。构建的pET41aEm18原核表达质粒，经IPTG诱导后，SDSPAGE检测表明rEm18GST重组蛋白得到成功表达，在相对分子量为50 KDa处有表达条带。结论: 成功克隆并构建了pET41aEm18原核表达质粒，初步诱导表达出rEm18GST重组蛋白，为新一代包虫病诊断试剂盒的研制奠定基础。

    关键词：泡球蚴； Em18基因；重组蛋白

    Cloning and Construction of pET41aEm18 prokaryotic expression plasmid and its primary expression in Escherichia coliWANG

    Yan, LIN Renyong, DING Jianbing, et al

    (Xinjiang Key Lab of Hydatid Fundamental Medical Research, First Affiliated Hospital,Xinjiang Medical University, Urumqi 830054, China)

    Abstract: Objective: To clone and construct the pET41aEm18 prokaryotic expression plasmid and to study its primary expression in Escherichia coli. Methods: The primers of Em18 were designed by DNAman biosoftware and the Em18 antigen cDNA was amplified by PCR from total RNA of Echinococcus multilocularis, and was cloned into prokaryotic expression plasmid pET41a to construct the pET41aEm18. The recombinant plasimid was transformed into E.coli BL21(DE3)and the positive clones were screened by restriction endonuclease analysis, and then sequenced. The sequences were analyzed by DNAman and GenBank/Blast biosoftware. The rEm18GST fusion protein was primary expressed by induction with IPTG and was detected by SDSPAGE. Results: DNA sequence analysis of Em18 cDNA fragment indicated that the length of Em18 was 486bp and code 161aa and was accepted by Genbank as a new sequence (AY513691). The pET41aEm18 positive clone was the exact recombinant plasmid and the rEm18GST recombinant protein was primary expressed as a band of 50 KDa by SDSPAGE detection. Conclusion: pET41aEm18 was constructed successfully and the rEm18GST recombinant protein was expressed primarily and has potential for use in the research of diagnosis for alveolar echinococcosis and the possible development of a diagnosis kit. ......

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