The Preliminary Study On The Structure And Expression Of Human Cytomegalovirus UL138,UL139 And UL140 Genes In Low Passage Clinical Isolates

Objetive: The purpose of our study is to research Human Cytomegalovirus (HCMV) UL138,UL139 and UL140 genetic structure in low passage clinical isolates by cotransfected with BAC-HCMV and its upregulator pcDNA-pp71, then detect the expression of their mRNA, setting up foundation for researching the relationship between the pathogenicity and the outcome of congenital HCMV infection, and providing more plentiful data for revealing the functions of the productions encoding by UL138, UL139,UL140 genes and the pathogenesis of congenital HCMV infection.Methods: HCMV strains were isolated from HELF cells which are cotransfected with BAC-HCMV plasmid and its upregulator pcDNA-pp71 ; collect the cells while the HELF cell appear cytopathic effect; the UL138, UL139 and UL140 genes primers were designed for complete coding region according to Toledo complete genomic provided in GeneBank, HCMV RNA was extracted from cells and the expression of UL138 and UL139,UL140 mRNA were detected by RT-PCR method , then cloned into pMD18-T vector and sequencd subsequently; use bioinformatics methods to analyse their genetic structure.Results: HCMV' s upregulator pcDNA-pp71 plasmid was successfully constructed ; The cotransfection of the HCMV BACs and pcDNA-pp71 plasmid into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes; The expression of HCMV UL138 and UL139 , UL140 mRNA were detected from the reconstituted infectious virus;The RT-PCR products were cloned into pMD18-T vector, These recombinant plasmids were subsequently sequenced and analyzed.