Construction and identification of the recombinant plasmid pET30a-EgA31-Eg95 of Echinococcus granulosus.
Construction and identification of the recombinant plasmid pET30a-EgA31-Eg95 of Echinococcus granulosus.
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细粒棘球绦虫重组质粒pET30a-EgA31-Eg95的构建及鉴定
DOI：
10.3892/etm.2013.1393
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发表时间：
2014-01
影响因子：
2.7
通讯作者：
Ding J
中科院分区：
文献类型：
--
作者：
Li Y;Zhang F;Althunayan MH;Hu XA;Xin Y;Jia H;Guo Y;Ma X;Wen H;Ding J
将Eg 95和EgA 31抗原基因克隆到原核表达质粒pET 30 a-EgA 31-Eg 95中，获得了重组蛋白EgA 31-Eg 95，并进行了Western blot分析。从细粒棘球绦虫（Echinococcus granulosus，E.颗粒虫）蠕虫。通过定量实时聚合酶链反应（qPCR）扩增编码EgA 31抗原的互补DNA（cDNA）。以重组质粒pET 30 a-EgA 31为载体，与Eg 95载体连接。构建重组质粒pET 30 a-EgA 31-Eg 95，经SDS-PAGE检测融合蛋白EgA 31-Eg 95的表达。阳性克隆为空重组载体。重组蛋白pET 30 a-EgA 31-Eg 95分子量约为46 kDa，表达产物约占可溶性蛋白的20%。成功构建了重组质粒pET 30 a-EgA 31-Eg 95，并在大肠杆菌中表达了重组蛋白EgA 31-Eg 95。为进一步研究其免疫原性奠定了基础。
To clone the Eg95 and EgA31 antigen genes into the prokaryotic expression plasmid pET30a-EgA31-Eg95, we expressed the recombinant protein EgA31-Eg95 and confirmed with western blot analysis. The total RNA was extracted from the protoscoleces of Echinococcus granulosus (E. granulosus) adult worms. The complementary DNA (cDNA) encoding the EgA31 antigen was amplified via quantitative real-time polymerase chain reaction (qPCR). The recombinant plasmid pET30a-EgA31 was used as a carrier and was connected with the Eg95 vector. The recombinant plasmid pET30a-EgA31-Eg95 was constructed and the fusion protein EgA31-Eg95 was detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The positive clone was the empty recombinant vector. The recombinant protein pET30a-EgA31-Eg95 was ~46 kDa, and the expressed product accounted for approximately 20% of the total soluble proteins. We successfully constructed the recombinant plasmid pET30a-EgA31-Eg95 and expressed the recombinant protein EgA31-Eg95. The results may be the foundation of research on its immunogenicity in the future.
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