Clone And Expression Of Human Cytomegalovirus Phosphoprotein 65 Gene In Chinese Hamster Ovary Cell

Objective To develop a gene engineered subunit vaccine of human cytomegalovirus (HCMV) against its infection, phosphoprotein 65 (pp65), a major antigen of HCMV, was expressed in Chinese hamster ovary (CHO) cell. Method HCMV strain AD169 was propagated in WI-38 cell and viral DNA was extracted as a template for PCR amplification of UL83 (pp65), the resulting PCR was subcloned into pUC118HincII/BAP plasmid and DNA sequence analysis conformed the fidelity of the PCR. The vector PcDNA5.0 was designed to correctly place CMV promoter sequence, pp65 sequence and secret signal sequence (mouse Ig kappachain for efficient secretion of recombination protein) into its genomic DNA. Exchanged primers of pp65 sequence, CMV promoter sequence and secret signal sequence to confirm the result by PCR screening. The vector PcDNAS.O was transfected into CHO cell, supernatants of transfected cells were extracted and purified. Recombination protein from supernatants was detected by gel electrophoresis, dot blot hybridization of Western- ECL system and Westen-bloting. Result 1.Compared the sequence of pp65 gene with the standard sequence of pp65 from Medline, we found that the concordant rate between them is 99.94%, only a nonsense mutation occurrences at 1455 base. 2.We have established a PcDNA5.0 Eukaryotic expression vector,which including CMV prmotor sequence , secretion signal sequence and pp65 sequence. PCR screening and the pp65 protein expressed in CHO cell confirmed it. 3.Extraction from supernatants in transfected CHO cells is recombination protein of pp65, which was detected by gel electrophoresis and dot blot hybridization of Western- ECL system and westen bloting. Conclusion 1. Gene cloning of HCMV UL83 (pp65 gene) was implementation.2.A transfer Eukaryotic expression vector PcDNA5.0 was constructed by CMV promoter sequence, secretion signal sequence and pp65 gene squence, it can be use as HCMV gene vaccine. 3.Recombination protein of HCMV pp65 gene has been expressed in CHO cell, Purification of it from the supernatants of CHO cells is the major antigen prepares for develops the subunit vaccine of HCMV.