Construction and identification of the recombinant plasmid pET30a-EgA31-Eg95 of Echinococcus granulosus.

ABSTRACT: To clone the Eg95 and EgA31 antigen genes into the prokaryotic expression plasmid pET30a-EgA31-Eg95, we expressed the recombinant protein EgA31-Eg95 and confirmed with western blot analysis. The total RNA was extracted from the protoscoleces of Echinococcus granulosus (E. granulosus) adult worms. The complementary DNA (cDNA) encoding the EgA31 antigen was amplified via quantitative real-time polymerase chain reaction (qPCR). The recombinant plasmid pET30a-EgA31 was used as a carrier and was connected with the Eg95 vector. The recombinant plasmid pET30a-EgA31-Eg95 was constructed and the fusion protein EgA31-Eg95 was detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The positive clone was the empty recombinant vector. The recombinant protein pET30a-EgA31-Eg95 was ~46 kDa, and the expressed product accounted for approximately 20% of the total soluble proteins. We successfully constructed the recombinant plasmid pET30a-EgA31-Eg95 and expressed the recombinant protein EgA31-Eg95. The results may be the foundation of research on its immunogenicity in the future.

SUBMITTER: Li Y 

PROVIDER: S-EPMC3861358 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature