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HCMV infection can cause hepatopathy,pneumonia,diarrhea,thrombocytopenic purpura,and sensorineural hearing loss(SNHL),with high morbility and mortality.pp65(phosphoprotein 65),a major constitutive protein of virion,is closely associated with viral gene expression,immune evasion and cell metabolism.pp65 antigenemia level has positive correlation with clinical symptom,which can guide the preemptive therapyand improve prognosis,is considered as one of the golden standard methods of active cytomegalovirus infection.In this paper,the pp65 protein properties,pathogenesis,application in diagnosis and prevention are reviewed.
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Objective: To investigate the potential role of human cytomegalovirus lower matrix phosphoprotein 65 (HCMV-pp65) in murine systemic lupus erythematosus (SLE). Methods: The prokaryotic plasmid pET-28b-pp65 was constructed to express the HCMVpp65 protein. BXSB mice and C57BL/6 mice were inoculated with pp65 eukaryotic plasmid pcDNA3.0-pp65 intramuscularly 5 times at 2-week intervals, and then the blood of the mice was subsequently collected via the retro-orbital vein. Indirect ELISAs were used to evaluate the concentration of anti-pp65 immunoglobulin G, anti-double-stranded DNA and antinuclear antibodies. Interleukin-1β and tumor necrosis factor-α were also determined by competitive ELISA. At the same time, 3 major SLE-related circulating microRNAs were examined by quantitative RT-PCR. Results: The early production of autoantibodies was observed in pp65-immunized male BXSB as well as C57BL/6 mice. Overexpression of interleukin-1β and tumor necrosis factor-α were detected in pp65-immunized male BXSB mice. Quantitative RT-PCR analyses showed that three SLE related microRNAs (microRNA-126, microRNA-125a, and microRNA-146a) were downregulated in peripheral blood mononuclear cells of pp65-immunized mice. Conclusions: Our findings indicate that HCMV-pp65 immunization strongly triggers the development and progression of SLE-like disease in both BXSB and C57BL/6 mice, which indicates that the immune responses induced by HCMV-pp65 may be involved in the development of SLE.
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Objective To investigate the potential role of human cytomegalovirus lower matrix phosphoprotein 65 (HCMV pp65) in murine systemic lupus erythematosus (SLE).Methods The prokaryotic vector pET-28b and eukaryotic vector pcDNA 3.0 were constructed to express the HCMV pp65 protein.All the C57BL/6 mice were inoculated with pp65 eukaryotic vector intramuscularly five times at 2-week intervals and then were bled via the retro-orbital vein.Subsequently,indirect ELISA was used to evaluate the concentration of anti-pp65 IgG,anti-dsDNA and ANA.At the same time,IL-1 b,IL-6,and TNF-α were determined by competitive ELISA.Results The early onset of autoantibodies and an overexpression of IL-6 were observed in immunized male C57BL/6 mice.Conclusion HCMV pp65 triggers the deregulation of humoral immunity in C57BL/6 mice,which indicates that the immune responses induced by HCMV pp65 may be involved in the development of SLE.
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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.
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0.05).Conclusion Three Hybridoma cell lines which secrete the target antibodies with satisfied affinities and specificities have been successfully raised,which provides a basis to produce a domestic-made HCMVpp65 antigen diagnosis kit.