| ObjectiveThe human cytomegalovirus (HCMV) infection is widespread in the crowd. HCMV infection can cause many diseases of nervous and digestive system, which has been one of important infection factor causing neonatal diseases and congenital malformation in children. However, the pathogenic mechanism is still uncertain. In 1996, Cha TA et al. compared nucleotide sequence of HCMV laboratory strains AD169, Towne with that of low-passage separates strain Toledo, and found that Toledo strain contained 19 new genes in UL/b'area that nonexistent in AD 169 strain, concluding HCMV UL133,UL134,UL135…L151. The 19 new genes may play an important role in replication of viruses, delitescence, and pathogenicity to host. In our study, by yeast two-hybrid system, We conducted a study of screening human fetal brain Lid protein factor interaction with HCMV UL135 protein, further searching for possible pathway that HCMV infection caused nervous system disease. Our study screened the target protein from fetal brain Lid by segments in the region as a bait protein for further in-depth study the pathogenic mechanism of HCMV basis.Materials and methods1.We extracted HCMV DNA from infected human cytomegalovirus in human fetal lung, amplified UL135 gene sequence by PCR technology to HCMV DNA as a template, then purified and connected after the UL135 PCR products with the vector pGBKT7 double digestion, last screened and identified recombinant clone (pGBKT7-UL135), sequenced and analyzed.2. We screened proteins that interact with the HCMV UL135 protein from human fetal brain cDNA Lid by yeast two-hybrid technique.(1) Extracting library plasmid; (2) Transforming recombinant plasmid contained pGBKT7-UL135 into AH109 yeast cells, then to transform library plasmid into the AH109 contained pGBKT7-UL135;(3) Screening positive clones (color reaction and PCR technology), identifying positive clones and sequencing the cDNA library which interact with HCMV-UL135;(4) BLAST analysis.Results1. We get HCMV UL135 gene fragment using specific primers, the fragment length of 987bp, and successfully cloned the HCMV UL135 into pGBKT7-binding domain of transcription of yeast, the named pGBKT7-UL135. We applicated PCR technology to identity positive clones containing pGBKT7-UL135, then extracted pGBKT7-UL135 plasmid, identification with EcoR I and BamH I restriction enzyme, and sequencing analysis showed that in line with expectations.2. Transforming library plasmids into the AH109 which contains pGBKT7-UL135 and determining conversion efficiency of yeast cells. In the Medium defect plate,260 clones grown, calculating conversion efficiency=0.26×10 cfu/μg. To do color reaction to screen the false positive clones, there are 95 blue colonies, and extract its yeast plasmid to amplify.3. Electroporating the extracted yeast plasmid into E. coli and identifying the transformed colonies (using Library of upstream and downstream primers to identify whether the transformation is from library information), results showed that positive clone DNA fragment size is 500bp-1500bp, and 60 positive clones were selected, and then will turn positive clones containing plasmid pGBKT7-UL135 genes in yeast cells AH 109. Sequencing and BLAST analysis showed there are multiple clones interact with the protein encoded by UL135, of which two clones highly homologous to Thy-1, the homology rate of 98%.ConclusionSome proteins interacting with HCMV UL135 in Human fetus brain cDNA Library were successfully screened and Thy-1 may play an important role in the course of HCMV infection.
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