摘要
目的从新疆株细粒棘球蚴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，蛋白表达及功能初步鉴定。方法设计特异性引物，从新疆株细粒棘球蚴中提取总RNA，RT—PCR法扩增EgERKl基因，构建pET28a—EgERK1原核表达质粒，测序确定序列并进行生物信息学分析。构建正确的pET28a-EgERK1原核表达质粒，经诱导、表达EgERK1重组蛋白，聚丙烯酰胺凝胶电泳和Western印迹检测其生物学功能。结果RT-PCR扩增出的条带经测序，结果显示其长度为1125bp，编码374个氨基酸，等电点为6．34，BLAST比对结果提示为一新基因，命名为EgERKl（EU701008）。同源性比较表明，EgERK1与多房棘球绦虫ERK基因（EmMPK1）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％。进化树分析发现EgERKl和EmMPKl相聚集。功能分析预测EgERKl具有ERK类激酶T—X—Y结构保守区和酶激活功能域。Western印迹显示，原核诱导表达的EgERKl重组蛋白能与抗人ERK1／2抗体发生特异性免疫反应。结论成功克隆细粒棘球蚴EgERKl新基因，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 To perform molecular cloning and sequencing, bioinformatics analysis, protein expression and function of extracellular signal regulated kinase （EgERK1） of Echinococcus granulosus in Xinjiang. Methods The specific primers of EgERK1 were designed and total RNA was extracted from Echinococcus granulosus in Xinjiang. EgERK1 gene was amplified by reverse transcription-polymerase chain reaction （RT-PCR） and prokaryotic expression plasmid pET28a- EgERK1 was constructed and sequenced. The sequences were analyzed by DNA sequencing and bioinformatics technology. The recombinant EgERK1 protein was induced and expressed. The biological function was detected using sodium dodecyl sulfate polyacrylamide gel electropheresis and Western blot. Results The sequence of RT-PCR product was 1125 bp, encoding 374 amino acids with isoelectric point of 6.34. This gene was a new ERK-homologues gene indicated by BLAST, named EgERK1 （EUT01008）. Homology comparisons indicated that the homology of EgERK1 and EmMPK1 from Echinococcus multilocularis was 95. 45%, and was43. 04%- 61. 88% to ERK from Caenorhahditis elegans, S. cerevisiae, D. melanogaster and human. Phylogenetic analysis showed that EgERK1 clustered with EmMPK1. Bioinformatics analysis predicted that EgERK1 contained a highly conserved T-X-Y motif and activation loop segment of ERK-like kinase. Western blot results showed tbe EgERK1 recombinant protein could reacted specifically with anti-human ERK monoelonal antibody. Conclusion A new EgERK1 gene of Echiuococcus granulosus is successfully cloned and its recombinant protein could reacted specifically with ERK1/2 antibody, which provides the basis for further study of EgERK1 function in the host-parasite interaction.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2010年第7期402-407,共6页
Chinese Journal of Infectious Diseases
基金
基金项目：国家自然科学基金资助项目（30760228、30860253）
关键词
细粒棘球绦虫
细胞外信号调节MAP激酶类
基因
克隆
分子
序列分析
质粒
Echinococcus granulosus
Extracellalar signal-regulatea MAP kinases
Gene
Cloning, molecular
Sequence analysis
Plasmids
