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A Study of Biological Function of Human Cytomegalovirus UL135 Protein

Author: ZouFei
Tutor: ZhouXiaoZuo
School: China Medical University
Course: Pediatrics
Keywords: Human cytomegalovirus UL135 protein Yeast two-hybrid system A human fetal brain cDNA library
CLC: R373
Type: Master's thesis
Year: 2010
Downloads: 29
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Abstract

Purpose of human cytomegalovirus (human cytomegalovirus HCMV) infection in the population prevalence of HCMV infection can cause nervous system and digestive system of children with a variety of diseases, has become an important infection caused by neonatal diseases and congenital malformations factor , but the pathogenic mechanisms of congenital HCMV infection is unclear. In 1996, Cha TA Towne strain and a laboratory strain of HCMV AD169 strain, named for Toledo low-passage isolate a nucleic acid sequence comparisons, found Toledo strains contained in UL / b 'District 19 does not exist in the AD169 strain The new gene, were named as HCMV UL133, UL134, UL135 ... UL151, 19 genes may play an important role in the replication of the virus, the latent and pathogenic host. In this study, the yeast two-hybrid system for protein factor means of filtering the HCMV UL135 protein interactions of human fetal brain cDNA library, and then find out the possible ways of HCMV infection can cause nervous system disease. As bait protein fragments of the research in this area, screened from the library of human fetal brain cells bind to the target protein, and to lay the foundation for further in-depth study of the pathogenic mechanisms of HCMV. Materials and methods, experimental materials specimens of the China Medical University Shengjing Hospital the virus Room save low passage clinical isolates of H; construct expression vectors related reagents; Yeast two-hybrid experiments related reagents; commonly used laboratory equipment such as low temperature BECKMAN Desktop high-speed centrifuge, Perkin Elmer PCR cycler, electroporation instrument (BioRad), temperature oscillation incubator; common database and analysis software, such as genomic databases, protein analysis database, gel imaging and scanning software, primer design software and so on. Second, the experimental methods extracted from human fetal lung infected with human cytomegalovirus HCMV DNA, the UL135 gene sequence was amplified PCR technique HCMV DNA as a template, UL135 amplification products with the vector pGBKT7 simultaneous dual digested purification, connection, screening of recombinant clones (pGBKT7-UL135), sequenced and analyzed. With HCMV UL135 protein encoded protein interactions in the yeast two-hybrid technology to screen a human fetal brain cDNA library. 1, extraction of the library plasmid; 2 containing pGBKT7-UL135 recombinant plasmid was transformed into yeast cells AH109, ??and then extracted library plasmids were transformed into the yeast cells AH109 containing pGBKT7-UL135 plasmid; positive clones (color reaction and PCR techniques), rotary identification, interaction with HCMV-UL135 cDNA gene sequencing; 4, BLAST analysis. HCMV UL135 gene fragment was amplified results using specific primers, and the fragment length of 987bp, and successfully the HCMV UL135 fragments cloned into the yeast system the transcription binding domain pGBKT7, pGBKT7-UL135. Technical appraisal of the application of PCR positive clones containing plasmid pGBKT7-UL135, and then extract pGBKT7-UL135 plasmid with EcoR I and BamH I restriction enzyme digestion identification, sequencing analysis showed results consistent with the expected. Conversion library plasmid to contain the pGBKT7-UL135 AH109 yeast cells and transformation efficiency was measured, 260 clones were grown in two missing plates, the calculated conversion efficiency = 0.26 × 104cfu/μg, meet the requirements. Do color reaction four missing screening to exclude false positive clones growth medium, 95 colonies were blue, and its value-added and extracted yeast plasmid. Amp (ampicillin)-containing tablet transformed colonies, PCR identification of the extracted yeast plasmid was transformed into E. coli after coating (with a library on the downstream primer, in order to identify the transformed whether the library information), the results show that the PCR method identified the size of the positive clone DNA fragments between 500bp-1500bp, selected positive clones 60, and then positive clones plasmid rotary yeast cells AH109 with pGBKT7-UL135 gene, were grown in the two short and four missing medium. Sequencing and BLAST analysis showed that the multiple cloning UL135 encoded protein interactions, two of which Cloning and Thy-1 (CD90) was highly homologous its the homologous rate of 98%. The conclusions of the successful application of the yeast two-hybrid system to filter out the protein encoded protein interactions with HCMV UL135 Thy-1, which may play an important role in HCMV infection pathogenic process.

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus)
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