The Preliminary Study On The Structure And Expression Of Human Cytomegalovirus UL144 And UL145 Genes In Low Passage Clinical Isolates

Objetive: The purpose of our study is to research UL144 and UL145 genetic polymorphisms in low passage clinical isolates and 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 UL144 and UL145 genes and the pathogenesis of congenital HCMV infection.Methods: HCMV strains were isolated from infants who infected HCMV congenitally, then inoculated to well growth HELF cell; collect the cells while the HELF cell appear cytopathic effect; extract HCMV genome from cells, the UL144 and UL145 genes primers were designed for complete coding region according to Toledo complete genomic provided in GeneBank and amplified by PCR method, then cloned into pMD18-T vector and sequencd subsequently ; use bioinformatics methods to analyse their genetic polymorphisms; HCMV RNA was extracted from cells and the expression of UL144 and UL145 mRNA were detected by RT-PCR method.Results:HCMV UL144 and UL145 genes were amplified successfully in low passage clinical isolates and cloned into pMD18-T vector ; These recombinant plasmids were subsequently sequenced and analyzed. Alignment comparison revealed that UL144 and UL145 sequence of various clinical isolates contained 98.5 ~ 99.2% and 97. 2 ~ 97. 8% of nucleoptide and 98. 6~99. 4% and 97. 8~98. 5% of amino acid sequence homologies compared with those of Toledo, respectively. The expression of HCMV UL144 and UL145 mRNA were detected from low passage clinical isolates.Conclusions:We succeed in isolating four HCMV strains in low passage clinical isolates, amplifying and cloning HCMV UL144 and UL145 genes; and detecting the expression of UL145 mRNA in the HELF for the first time .