| M122蛋白与宿主因子Myst4的相互作用位点 |
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| 引用本文： | 王慧,张菊,舒赛男,刘兴楼,李革,方峰.M122蛋白与宿主因子Myst4的相互作用位点[J].实用儿科临床杂志,2011,26(22). |
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| 作者姓名： | 王慧 张菊 舒赛男 刘兴楼 李革 方峰 |
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| 作者单位： | 华中科技大学同济医学院附属同济医院儿科,武汉,430030 |
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| 基金项目： | 国家自然科学基金(30671859) |
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| 摘 要： | 目的 明确M122蛋白与宿主因子Myst4相互作用的位点.方法 以诱饵载体pGBKT7-M122为模板,采用PCR方法扩增不同长度大小的M122基因片段,并将其分别插入至pMD-T simple载体,构建含有不同长度M122基因片段的重组质粒.将构建成功的各个重组质粒分别用限制性内切酶EcoRI和SalI进行双酶切鉴定,并送测序.将测序正确的重组质粒采用同样的内切酶进行双酶切,凝胶回收试剂盒回收不同长度大小的M122基因片段,并将其分别亚克隆至pGBKT7-BD载体构建含有不同长度M122基因片段的诱饵质粒.将构建成功的各个诱饵质粒分别用限制性内切酶EcoRI和SalI进行双酶切鉴定,并送测序.将测序正确的各个诱饵质粒分别与pGADT7 -Myst4质粒共同转化至酵母菌株AH109感受态细胞,转化后的酵母细胞分别涂板于营养缺陷培养基SD/-Trp/-Leu和SD/-Trp/-Leu/-His/-Ade/X-α-Gal平板.M122与宿主因子Myst4相互作用的位点通过营养缺陷筛选确定.结果 成功扩增了编码不同M122蛋白突变体的基因片段,并将其正确插入诱饵载体,构建了含有不同长度大小M122基因片段的诱饵质粒；通过酵母双杂交筛选明确了M122蛋白与宿主因子Myst4相互作用的结合位点位于M122蛋白的1～ 148氨基酸.结论 M122蛋白的1～ 148氨基酸是其与宿主因子Myst4相互作用所必需的,为研究M122蛋白在MCMV致神经系统损伤的分子机制中的作用提供了实验基础.
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| 关 键 词： | 巨细胞病毒 M122蛋白 Myst4 酵母双杂交分析 |
Interaction Sites between M122 Protein and Host Factor Myst4 |
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| WANG Hui
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ZHANG Ju
,
SHU Sai-nan
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LIU Xing-lou
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LI Ge
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FANG Feng.Interaction Sites between M122 Protein and Host Factor Myst4[J].Journal of Applied Clinical Pediatrics,2011,26(22). |
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| Authors: | WANG Hui ZHANG Ju SHU Sai-nan LIU Xing-lou LI Ge FANG Feng |
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| Institution: | WANG Hui,ZHANG Ju,SHU Sai-nan,LIU Xing-lou,LI Ge,FANG Feng(Department of Pediatrics,Tongji Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,Hubei Province,China) |
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| Abstract: | Objective To find the domains of M122 involved in the interaction with Myst4. Methods The DNA fragments encoding various truncated M122 were amplified by polymerase chain reaction using pGBKT7-M122 as a template,and then inserted into pMD-T smiple vector,respectively.After verified with restriction endonuclease digestion of EcoRI and SalI,the right fragments of various truncated M122 gene determined by sequencing were subcloned into pGBKT7-BD vector,respectively.All the recombinant plasmids were verified by... |
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| Keywords: | cytomegalovirus M122 protein Myst4 yeast two-hybrid assay |
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