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目的:利用酵母双杂交系统从人胎脑cDNA文库中筛选与人巨细胞病毒(HCMV)UL131A编码蛋白相互作用的蛋白.方法:将成功构建的酵母诱饵表达载体pGB-KT7-UL131A转化到酵母菌AH109中,再将人胎脑文库DNA转化到含有pGBKT7-UL131A的酵母细胞中,筛选与HCMV UL131A编码蛋白相互作用的细胞蛋白,并对筛选得到的阳性克隆进行测序和生物信息学分析.结果:成功构建酵母诱饵表达载体pGBKT7-UL131A,并将其与人胎脑cDNA文库共转化到酵母细胞AH109中,最终确认有23种人胎脑文库蛋白与HCMV UL131A编码蛋白相互作用,其中一种与Thy-1蛋白高度同源,其同源性高达99%.结论:成功应用酵母双杂交系统筛选出与HCMV UL131A编码蛋白相互作用的蛋白,其中Thy-1在HCMV感染致病过程中可能起重要作用.
OBJECTIVE: To screen the human fetal brain cDNA library for interaction with human cytomegalovirus (HCMV) UL131A protein by yeast two-hybrid system.Methods: The yeast bait expression vector pGB-KT7-UL131A was successfully transformed into yeast AH109, the human fetal brain DNA was transformed into yeast cells containing pGBKT7-UL131A to screen for cellular proteins that interact with HCMV UL131A-encoding proteins, and the positive clones were sequenced and bioinformatics analyzed.Results: The yeast bait expression vector pGBKT7-UL131A was successfully constructed and co-transformed into human fetal brain cDNA library into yeast cell AH109. Twenty-three human fetal brain library proteins were confirmed to interact with the HCMV UL131A-encoding protein. One of them interacts with Thy 1 protein was highly homologous and its homology was as high as 99% .Conclusion: The yeast two-hybrid system was successfully used to screen out the protein interacting with HCMV UL131A protein, and Thy-1 may play an important role in the pathogenesis of HCMV infection .