Development of A Gene Vaccine Phosphoprotein 65 of Human Cytomegalo Virus / 实用儿科临床杂志

ABSTRACT

Objective To explore the method to develop a gene vaccine of human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) against its infection. Methods HCMV strain AD169 was propagated in WI-38 cell and viral DNA was extracted as a template for polymerase chain reaction (PCR) amplification of UL83 (pp65), the resulting of PCR was subcloned into pUC118HincII/BAP plasmid and DNA sequence analysis conformed the fidelity of the PCR. The vector pcDNA 5.0 was designed to correctly place CMV promoter sequence, pp65 sequence and secret signal sequence (mouse immunoglobulin 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 pcDNA 5.0 was transfected into CHO cell, supernatants of transfected cells were extracted and purified. Recombination protein from supernatants was detected by gel electrophoresis and dot blot hybridization of Western- ECL system. Results Compared the sequence of pp65 gene with the standard sequence of pp65 from Medline,it was found that the concordant rate between them was 99.99%,only a nonsense mutation occurrences at 1 455 base.A pcDNA 5.0 Eukaryotic expression vector was established, which including CMV promoter sequence,secretion signal sequence and pp65 sequence. PCR screening and the pp65 protein expressed in CHO cell confirmed it. Extraction from supernatants in transfected CHO cells was recombination protein of pp65, which was detected by gel electrophoresis and dot blot hybridization of Western- ECL system and western blotting.Conclusion Subunit vaccine of HCMV is gained,which is a transfer eukaryotic expression vector pcDNA 5.0 constructed by CMV promoter sequence,secretion signal sequence and pp65 gene sequence.