Molecular cloning, sequencing and function of extracellular signal regulated kinase of Echinococcus granulosus
10.3760/cma.j.issn.1000-6680.2010.07.006
- VernacularTitle:细粒棘球蚴细胞外信号调节激酶基因克隆、序列分析及功能的初步鉴定
- Author:
Guodong Lü
;
Jing JI
;
Junhua WANG
;
Liang LI
;
Hongli WANG
;
Xiaomei LU
;
Xing WANG
;
Hao WEN
;
Renyong LIN
- Publication Type:Journal Article
- Keywords:
Echinococcus granulosus;
Extracellalar signal-regulatea MAP kinases;
Gene;
Cloning,molecular;
Sequence analysis;
Plasmids
- From:
Chinese Journal of Infectious Diseases
2010;28(7):402-407
- CountryChina
- Language:Chinese
-
Abstract:
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(EU701008).Homology comparisons indicated that the homology of EgERK1 and EmMPK1from Echinococcus multilocularis was 95.45%, and was 43.04%-61.88% to ERK from Caenorhabditis 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 the EgERK1 recombinant protein could reacted specifically with anti-human ERK monoclonal antibody. Conclusion A new EgERK1 gene of Echinococcus 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.
