細(xì)粒棘球蚴細(xì)胞外信號調(diào)節(jié)激酶（EgERK1）基因的克
發(fā)布時間：2018-11-27 20:17
【摘要】： 目的:從細(xì)粒棘蚴(Echinococcus granulosus,Eg)中克隆細(xì)胞外信號調(diào)節(jié)激酶(EgERK1)基因,進(jìn)行序列測定,生物信息學(xué)分析,構(gòu)建pET28a-EgERK1原核表達(dá)質(zhì)粒,經(jīng)誘導(dǎo)、表達(dá)并純化重組rEgERK1,Western Blot檢測rEgERK1重組蛋白生物學(xué)特性,為進(jìn)一步研究該基因在寄生蟲與宿主相互作用中的功能奠定基礎(chǔ)。方法:設(shè)計EgERK1基因特異性引物,從新疆株細(xì)粒棘球蚴中提取總RNA,RT-PCR法擴(kuò)增EgERK1基因,構(gòu)建pMD19-T/EgERK1質(zhì)粒,測序確定序列并進(jìn)行生物信息學(xué)分析。構(gòu)建pET28a-EgERK1原核表達(dá)質(zhì)粒,測序鑒定插入序列正確性。IPTG誘導(dǎo)表達(dá)rEgERK1-His重組蛋白,Ni-NTA His Bind Resin親合層析柱純化,SDS-PAGE法確定蛋白表達(dá)情況,Western Blot檢測其生物學(xué)功能。結(jié)果:RT-PCR擴(kuò)增出一條長度為1100bp的條帶,測序結(jié)果顯示其長度為1125bp,編碼374個氨基酸,等電點(diǎn)為6.34,為一新基因,命名為EgERK1(EU701008)。同源性比較表明EgERK1與多房棘球絳蟲EmMPK1基因同源性為95.45 %,與線蟲、酵母、果蠅和人類等ERK基因的同源性為43.04～61.88 %。進(jìn)化樹分析結(jié)果發(fā)現(xiàn)EgERK1和多房擊球絳蟲ERK基因(EmMPK1)相聚集。功能分析預(yù)測EgERK1具有ERK類激酶T-X-Y結(jié)構(gòu)保守區(qū)和酶激活功能域。成功構(gòu)建了pET28a-EgERK1原核表達(dá)質(zhì)粒,經(jīng)IPTG誘導(dǎo),SDS-PAGE檢測表明rEgERK1-His重組蛋白得到成功表達(dá),在相對分子量47KDa處有表達(dá)條帶;Western Blot分析顯示rEgERK1-His重組蛋白能被特異性抗人ERK1/2單克隆抗體識別。結(jié)論:首次克隆細(xì)粒棘球蚴EgERK1新基因,成功構(gòu)建高效融合表達(dá)基因工程菌株pET28a-EgERK1,成功誘導(dǎo)表達(dá)并純化EgERK1重組蛋白,發(fā)現(xiàn)EgERK1重組蛋白具有與ERK1/2抗體結(jié)合的功能,為進(jìn)一步研究該基因在寄生蟲與宿主相互作用中的功能奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R346
本文編號：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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R346
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