细粒棘球蚴细胞外信号调节激酶（EgERK1）基因的克隆、序列分析及功能的初步鉴定
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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。
Objective:Molecular cloning and characterization of EgERK1 from protoscolex of Echinococcus granulosus (E. granulosus) in Xinjiang. Methods: In accordance with the EmMPK1 sequence, the primers of EgERK1 were designed. Total RNA was extracted from protoscoles of E. granulosus and EgERK1 was amplified by RT- PCR and then cloned to be pET28a-EgERK1 for sequencing. The sequences were analyzed by DNAMAN and BLAST and recombinant protein EgERK1 was detected by Western Blot. Results: The new ERK- homologues genes, named EgERK1 (EU701008), was cloned from protoscoles and DNA sequence showed EgERK1 include 1125bp, coding 374aa, PI was 6.34 and has 95.45 % homology to EmMPK1 and 43.04-61.88 % homology to other species. Phylogenetic analysis indicated that EgERK1clustered with EmMPK1.Bioinformatics analysis showed that EgERK1 contains an ERK protein highly conserved T-X-Y motif and its activation loop segment is same as other MAPK kinases. The Western blot result showed that the EgERK1 recombinant protein could specific reacted with anti-human ERK monoclonal antibody. Conclusion: A new ERK gene, EgERK1, was cloned from protoscolex of E. granulosu and could reacted with ERK antibody, which provides the basis for further study of EgERK1 expression and functions between the host and parasite.
Objective:Molecular cloning and characterization of EgERK1 from protoscolex of Echinococcus granulosus (E. granulosus) in Xinjiang. Methods: In accordance with the EmMPK1 sequence, the primers of EgERK1 were designed. Total RNA was extracted from protoscoles of E. granulosus and EgERK1 was amplified by RT- PCR and then cloned to be pET28a-EgERK1 for sequencing. The sequences were analyzed by DNAMAN and BLAST and recombinant protein EgERK1 was detected by Western Blot. Results: The new ERK- homologues genes, named EgERK1 (EU701008), was cloned from protoscoles and DNA sequence showed EgERK1 include 1125bp, coding 374aa, PI was 6.34 and has 95.45 % homology to EmMPK1 and 43.04-61.88 % homology to other species. Phylogenetic analysis indicated that EgERK1clustered with EmMPK1.Bioinformatics analysis showed that EgERK1 contains an ERK protein highly conserved T-X-Y motif and its activation loop segment is same as other MAPK kinases. The Western blot result showed that the EgERK1 recombinant protein could specific reacted with anti-human ERK monoclonal antibody. Conclusion: A new ERK gene, EgERK1, was cloned from protoscolex of E. granulosu and could reacted with ERK antibody, which provides the basis for further study of EgERK1 expression and functions between the host and parasite.
引文
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[19]曾宪旭,关剑,韩艳春.p-Bad112/136、p-Akt、p-Erk在乳腺癌癌变过程中的表达及意义[J].中国肿瘤临床, 2006 (33 ):738-742.
[20] Schüβler P, Grevelding CG, Kunz W. Identification of Ras,MAP kinases, and a GAP protein in Schistosoma mansoni by immunoblotting and their putative involvement in male–female interaction[J]. Parasitology,1997,115: 629-634.
[21] Boumediene B,Fnu N,Mahalia S,et al. Cell Cycle Regulatory Proteins in the Liver in Murine Trypanosoma cruzi Infection[J]. Landes Bioscience,2006,5:2396-2400.
[22]孙新.弓形虫速殖子在宿主细胞内增殖与丝裂原激活蛋白质激酶表达[J].蚌埠医学院学报,2002,2(7 4):283- 284.
[23]杨小迪,陈兴,智孙新.丝裂原蛋白激酶抑制剂U0126对刚地弓形虫侵入宿主细胞的影响[J].热带病与寄生虫学,2006,4:5-7.
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