细粒棘球蚴细胞外信号调节激酶（EgERK1）基因的克
发布时间：2018-11-27 20:17
【摘要】： 目的:从细粒棘蚴(Echinococcus granulosus,Eg)中克隆细胞外信号调节激酶(EgERK1)基因,进行序列测定,生物信息学分析,构建pET28a-EgERK1原核表达质粒,经诱导、表达并纯化重组rEgERK1,Western Blot检测rEgERK1重组蛋白生物学特性,为进一步研究该基因在寄生虫与宿主相互作用中的功能奠定基础。方法:设计EgERK1基因特异性引物,从新疆株细粒棘球蚴中提取总RNA,RT-PCR法扩增EgERK1基因,构建pMD19-T/EgERK1质粒,测序确定序列并进行生物信息学分析。构建pET28a-EgERK1原核表达质粒,测序鉴定插入序列正确性。IPTG诱导表达rEgERK1-His重组蛋白,Ni-NTA His Bind Resin亲合层析柱纯化,SDS-PAGE法确定蛋白表达情况,Western Blot检测其生物学功能。结果:RT-PCR扩增出一条长度为1100bp的条带,测序结果显示其长度为1125bp,编码374个氨基酸,等电点为6.34,为一新基因,命名为EgERK1(EU701008)。同源性比较表明EgERK1与多房棘球绦虫EmMPK1基因同源性为95.45 %,与线虫、酵母、果蝇和人类等ERK基因的同源性为43.04～61.88 %。进化树分析结果发现EgERK1和多房击球绦虫ERK基因(EmMPK1)相聚集。功能分析预测EgERK1具有ERK类激酶T-X-Y结构保守区和酶激活功能域。成功构建了pET28a-EgERK1原核表达质粒,经IPTG诱导,SDS-PAGE检测表明rEgERK1-His重组蛋白得到成功表达,在相对分子量47KDa处有表达条带;Western Blot分析显示rEgERK1-His重组蛋白能被特异性抗人ERK1/2单克隆抗体识别。结论:首次克隆细粒棘球蚴EgERK1新基因,成功构建高效融合表达基因工程菌株pET28a-EgERK1,成功诱导表达并纯化EgERK1重组蛋白,发现EgERK1重组蛋白具有与ERK1/2抗体结合的功能,为进一步研究该基因在寄生虫与宿主相互作用中的功能奠定基础。
[Abstract]:Objective: to clone extracellular signal-regulated kinase (EgERK1) gene from hydatid granulosus (Echinococcus granulosus,Eg), sequence analysis, bioinformatics analysis, construct prokaryotic expression plasmid of pET28a-EgERK1, induce, express and purify recombinant rEgERK1,. The biological characteristics of rEgERK1 recombinant protein were detected by Western Blot, which laid a foundation for further study on the function of the gene in the interaction between parasite and host. Methods: the specific primers of EgERK1 gene were designed. The EgERK1 gene was amplified by total RNA,RT-PCR from Echinococcus granulosus of Xinjiang strain. The pMD19-T/EgERK1 plasmid was constructed and sequenced and analyzed by bioinformatics. The prokaryotic expression plasmid of pET28a-EgERK1 was constructed and the inserted sequence was confirmed by sequencing. The recombinant rEgERK1-His protein was induced by IPTG and purified by Ni-NTA His Bind Resin affinity chromatography. The expression of the protein was determined by SDS-PAGE method and its biological function was determined by, Western Blot. Results: a band of 1100bp was amplified by RT-PCR. The length of the band was 1125 BP, encoding 374 amino acids, and the isoelectric point was 6.34. It was a new gene named EgERK1 (EU701008). The homology between EgERK1 and EmMPK1 gene of Echinococcus multilocularis was 95.45%, and the homology with ERK gene of nematode, yeast, Drosophila and human was 43.04 (61.88%). The results of phylogenetic tree analysis showed that EgERK1 and ERK gene (EmMPK1) of Taenia multilocularis were clustered. Functional analysis predicted that EgERK1 had conserved domain of ERK kinase-like T-X-Y structure and functional domain of enzyme activation. The prokaryotic expression plasmid of pET28a-EgERK1 was successfully constructed. After IPTG induction, SDS-PAGE analysis showed that the recombinant rEgERK1-His protein was successfully expressed and expressed at the relative molecular weight of 47KDa. Western Blot analysis showed that rEgERK1-His recombinant protein could be recognized by specific anti-human ERK1/2 monoclonal antibody. Conclusion: the EgERK1 gene of Echinococcus granulosus was cloned for the first time, and the recombinant EgERK1 protein was successfully induced and purified by highly efficient fusion expression strain pET28a-EgERK1,. It was found that the recombinant protein of EgERK1 could bind to ERK1/2 antibody. It provides a basis for further study on the function of the gene in parasite-host interaction.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346
本文编号：2361919
[Abstract]:Objective: to clone extracellular signal-regulated kinase (EgERK1) gene from hydatid granulosus (Echinococcus granulosus,Eg), sequence analysis, bioinformatics analysis, construct prokaryotic expression plasmid of pET28a-EgERK1, induce, express and purify recombinant rEgERK1,. The biological characteristics of rEgERK1 recombinant protein were detected by Western Blot, which laid a foundation for further study on the function of the gene in the interaction between parasite and host. Methods: the specific primers of EgERK1 gene were designed. The EgERK1 gene was amplified by total RNA,RT-PCR from Echinococcus granulosus of Xinjiang strain. The pMD19-T/EgERK1 plasmid was constructed and sequenced and analyzed by bioinformatics. The prokaryotic expression plasmid of pET28a-EgERK1 was constructed and the inserted sequence was confirmed by sequencing. The recombinant rEgERK1-His protein was induced by IPTG and purified by Ni-NTA His Bind Resin affinity chromatography. The expression of the protein was determined by SDS-PAGE method and its biological function was determined by, Western Blot. Results: a band of 1100bp was amplified by RT-PCR. The length of the band was 1125 BP, encoding 374 amino acids, and the isoelectric point was 6.34. It was a new gene named EgERK1 (EU701008). The homology between EgERK1 and EmMPK1 gene of Echinococcus multilocularis was 95.45%, and the homology with ERK gene of nematode, yeast, Drosophila and human was 43.04 (61.88%). The results of phylogenetic tree analysis showed that EgERK1 and ERK gene (EmMPK1) of Taenia multilocularis were clustered. Functional analysis predicted that EgERK1 had conserved domain of ERK kinase-like T-X-Y structure and functional domain of enzyme activation. The prokaryotic expression plasmid of pET28a-EgERK1 was successfully constructed. After IPTG induction, SDS-PAGE analysis showed that the recombinant rEgERK1-His protein was successfully expressed and expressed at the relative molecular weight of 47KDa. Western Blot analysis showed that rEgERK1-His recombinant protein could be recognized by specific anti-human ERK1/2 monoclonal antibody. Conclusion: the EgERK1 gene of Echinococcus granulosus was cloned for the first time, and the recombinant EgERK1 protein was successfully induced and purified by highly efficient fusion expression strain pET28a-EgERK1,. It was found that the recombinant protein of EgERK1 could bind to ERK1/2 antibody. It provides a basis for further study on the function of the gene in parasite-host interaction.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346
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