| Objective:Molecular cloning and characterization of MKK1 and MKK1 from protoscolex of Echinococcus granulosus(E.granulosus)in Xinjiang.To construct the Echinococcos granulosus(Eg)14-3-3 and MKK2 in yeast two-hybrid system,and test the bait vector for transcriptional activation and toxicity.Methods:In accordance with the EmMKK1 and Em14-3-3 sequence,the primers of EgMKK1 and Eg14-3-3 were designed.Total RNA was extracted from protoscoles of E.granulosus and EgMKK1 and Egl4-3-3 were amplified by RT-PCR and then cloned to be pET41a-EgMKK1 and pET41a-Egl4-3-3 for sequencing.The sequences were analyzed by DNAMAN and BLAST and recombinant protein EgMKKl and Eg 14-3-3 were detected by Western Blot.The complete encoding gene of Eg 14-3-3 and EgMKK2 by PCR and they were then subcloned into pGADT7 and pGBKT7 of yeast two-hybrid vectors respectively.Results:The new MKK1 homologues genes,named EgERMKKl(GenBank:JN573355.1),was cloned from protoscoles and DNA sequence showed EgMKK1 include 1017bp,coding 338aa,PI was 6.47and has 98.7%homology to EmMKK1.The 14-3-3 homologues genes,named Eg14-3-3 was cloned from protoscoles and DNA sequence showed Eg14-3-3 include 742bp,coding 246aa,PI was 4.80 and has 97.30%homology to Eml4-3-3.And the complete encoding genes ofEgMKK2 were 1572bp respectively.Phylogenetic analysis indicated that EgMKKl clustered with EmMEK1.The Western blot result showed that the EgMKK1 recombinant protein could specific reacted with anti-human MEK3/6 monoclonal antibody.The pGBKT7-Eg14-3-3 and pGADT7-EgMKK2 vectors were constructed and identified that it had no toxicity and autonomous activation.Conclusion:A new MKK gene,EgMKK1,was cloned from protoscolex of E.granulosu and could reacted with MKK3/6 antibody,which provides the basis for further study of EgMKK1 expression and functions between the host and parasite.And layed the foundations for screening the Eg 14-3-3 and EgMKK2-interacting proteins using the yeast two-hybrid technique. |
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