Prokaryotic Expression And Preliminary Application Of Human Cytomegalovirus Pp65Gene In Prokaryotic System

Human cytomegalovirus (HCMV) is the largest member of the family of human herpesviruses. It is a widespread pathogen which is responsible for generally asymptomatic and persistent infections in healthy people. However it may cause severe disease in immunocompromised individuals. It easily spreads from pregnant women to their fetus through vertical transmission, resulting in fetal malformations, mental retardation and nervous system abnormalities. So it is of great significance to prenatal detection of HCMV infection in pregnant women. Enzyme-linked immunosorbent assay (ELISA) is a most commonly used method of the detection of HCMV antibody. However, many commercial ELISA kits based on recombinant antigen prepared. In order to improve the sensitivity, specificity and accuracy of HCMV antibody detection, the recombinant protein had been recommended to replace the whole-virus antigen for diagnosing HCMV infection. The membrane phosphoprotein pp65of HCMV contain T cell epitopes and B cell epitopes,which can induce neutralizing antibodies. The recombinant protein pp65will be expressed by recombinant DNA technology and it can be used as diagnostic antigen of HCMV infection.Objective:To prepare recombinant protein HCMV pp65by recombinant DNA technology for immunological diagnosis.Methods:According to the sequence of HCMV pp65gene in GeneBank, we designed a pair of primers, which were used for the amplified gene fragment by polymerase chain reaction (PCR), The purification PCR production and plasmid vector pGEX-4T-1DNA were digested with EcoRI and BamHI, then insert PCR production into plasmid vector to construct the recombinant plasmid, and identificate it. The recombinant plasmid pGEX-4T-1/pp65was transformed into BL21, then it is induced by IPTG for expression, the production was identificated by SDS-PAGE. The fusion protein of the supernatant was purified by affinity chromatography, the character of GST/pp65protein was identificated by indirect ELISA. The GST/pp65protein was used for replacing antigen to detect the antibody in human serum.Results:The successful amplification of pp65gene by PCR; The recombinant plasmid pGEX-4T-1/pp65was successfully constructed; The successful expression of GST/pp65recombinant protein; The GST/pp65recombinant protein has the characteristics of specificity.Conclusion:The recombinant protein GST/pp65can be used as diagnostic antigen of HCMV infection.