Cloning Of Gag Gene And Its Expression In E.coli Of Porcine Endogenous Retrovirus Derived From Chinese Experimental Miniature Pigs

The primers used to amply gag gene of Porcine Endogenous Retrovirus (PERV) derived from Chinese Experimental Miniature Pigs(CEMP) were designed and synthesized referring to the gag gene of PERV-PK15. The PERV gag gene was obtained by reverse-transcription polymerase chain reaction (RT-PCR) from RNA template derived peripheral blood lymphocyte of Chinese Experimental Miniature Pigs, cloned into the vector pGEM-T, to construct plasmids pGEM-gag. Sequence analysis reveals that the PERV gag gene of miniature swine of China consisted of 1 575bp, encoding 524 ammo acids, and that it is a highly conserved gene with only three nucleotide mutation sites compared to data published.The recombinant plasmids pGEM-gag was digested with Not I and Sal I, and the resulting fragment was inserted into the Not I and Sal I sites of the pGEX-4T-3 under the downstream sequences which named PGEX-gag Expression Vector. E.Coli component host BL21 was transformed with the PGEX-gag. The recombinant bacteria could express the gag gene after induced by IPTG, and the recombinant protein were analyzed on SDS-PAGE and stained with Comassbrilliant blue or detected by Western Blot analysis with monoclonal antibody against GST. The expression products of gag gene which fused with GST was 86KDa, corresponding to the estimated molecular size of the gag protein. The expression level of the recombinant protein was about 18% of the total bacterial proteins. After cell lysis and SDS-PAGE analysis, we found all the expression products exist in forms of inclusion body. After solubilization in the presence of strong denaturants, we achieved refolding of the denatured proteins by renaturation. The fusion protein was purified through CM Sepharose Fast Flow IonExchange Chromatography.Purified fusion protein was specific and sensitive as the antigen of indirect ELISA. Using recombinant protein as antigen, the ELISA for examining PERV antibodies was established. Polyclonal antiserum was prepared by the repeated immunization of rabbits with purified gag protein. Gag-specific antiserum was produced and verified by indirect ELISA .The rabbit polyclonal antibody titer was 1:32 000. The results show that GST-gag was expression stably in E.coli and this recombined protein has strong antigenicity, which can be used to develop the genetic engineering diagnostic reagent.