Prokaryotic Expression Of Human Cytomegalovirus Pp150 Gene And Its Antigenicity Analysis

Objective To construct Human Cytomegalovirus (HCMV) pp150 gene prokaryotic expression clones, for the preparation of efficient recombinant antigen for serological diagnosis of HCMV infection.Methods A pair of PCR primers were created from the pp150 gene of HCMV AD 169 strain and two restriction sites (EcoRJ and Hindll) were synthesized as part of the primers . A fragment coding for pplSO antigen epitopes (840-1048aa) was amplified by PCR technique . The target gene was inserted into the prokaryotic expression plasmid pMAL-P2 and pET-28a respectively , after the transformation of E. coll. , the expressed fusion protein was induced with IPTG and the expression levels were evaluated by SDS-PAGE . The fusion protein was purified by one-step MBP affinity resin and its antigenicity was identified by Western blot , employing 15 HCMV IgM positive sera . Also the ELISA coating with the recombinant protein was used to screen 236 sera of health pregnancy women.Results A 630bp fragment was amplified by PCR . The recombinant expression plasmid pMAL-P2-pp!50 and pET-28a-pp150 were proved to correct with the double digestion of EcoRI and Hindll respectively. The pMAL-P2-pp150 plasmid could express a 64KDa fusion protein efficirntly in K coli. , it account for approximately 10% Of total bacterial protein , but the pET-28a-pp150 was failed . The purified fusionprotein was in the position of 64Kda, it could identified 80% HCMV IgM positive sera by Western blot, the ELISA coating with the recombinant protein has a good agreement(96%) with the ELISA kit from whole virion antigens produced by our lab.Conclusion The pp150 fusion proteins expressed successfully in E.coli. remaining good antigenicity. It will be used in the serological diagnosis for acute and/or active HCMV infection after further perfected.