Cloning and expression of E2 gene of eastern equine encephalomyelitis virus

Objective:To clone and express the E2 gene of eastern equine encephalomyelitis virus(EEEV) in order to study the antigenicity of recombinant protein and to lay a foundation for development of genetic engineering diagnostic reagents.Methods:The E2 gene of EEEV was amplified from total RNA of infected mouse brain by RT_PCR and then cloned into pGEM_T easy vector. After DNA sequencing, the E2 gene fragment was cloned into GST fusion protein expression vector pGEX_5x_2 and expressed in E.coli . The antigenicity of recombinant protein was identified by Western_blotting and ELISA. Results:After insertion of E2 gene into the prokaryotic fusion protein expression vector pGEX_5x_2, the fusion protein GST_E2 was expressed in E.coli DH5a and its amount was about 24.5% of total bacterial protein. GST_E2 protein was obtained from inclusion body separation and washed with Triton X_100, 1*!mol/L and 2*!mol/L urea. GST_E2 was demonstrated to have good antigenicity by Western_blotting and indirect ELISA.Conclusions: GST_E2 was expressed stably in E.coli and this recombinant protein has strong antigenicity which can be used to develop the genetic engineering diagnostic reagent.