应用白细胞cDNA文库的酵母双杂交技术筛选HCTP4结合蛋白基因

Screening of HCTP4 interacting proteins in leukocytes by yeast-two hybrid technique

目的:用酵母双杂交技术筛选白细胞中与HCTP4蛋白结合的蛋白的编码基因. 方法:用多聚酶链反应(PCR)法扩增HCTP4基因,连接入酵母表达载体pGBKT-7中构建诱饵质粒,转化酵母细胞AH109并在其内表达,然后与转化了人白细胞文库质粒的酵母细胞Y187进行配合,在营养缺陷型培养基上进行双重筛选阳性菌落,增菌后提出质粒,转化入大肠杆菌(DH5α), 提出质粒并测序,进行生物信息学分析. 结果:成功克隆出HCTP4基因并在酵母细胞中表达,配合后选出在四缺(SD/-Trp-Leu-Ade-His)培养基及铺有X-α- 半乳糖(X-α-gal)的四重缺陷培养基上均能生长并变成蓝色的真阳性菌落44个,其中25个免疫球蛋白λ轻链:6个来自RP11-189K21克隆的人DNA序列;4个来自RP11- 507C10克隆的人DNA序列;2个人BAC克隆RPCI11- 75L1;1个人BAC克隆RP11-21M10;1个人遍素酶智能弹(MIB);1个来自RP11-867O8克隆的人DNA基因;1个来自RP3-509119克隆的人DNA序列;1个小核糖核蛋白G: 1个UMP-CMP激酶;1个新基因. 结论:成功克隆出HCTP4的结合蛋白,为进一步研究丙型肝炎病毒(HCV)的作用提供了新线索.

AIM: To investigate the biological function of HCTP4, yeast-two hybrid was performed to screen proteins interacting with HCTP4 in leukocytes. METHODS: The HCTP4 gene was amplified by polymerase chain reaction (PCR) and HCTP4 bait plasmid was constructed by using yeast-two hybrid system 3, then the constructed vector was transformed into yeast AH109. The transformed yeast mated with yeast Y187 containing leukocytes cDNA library plasmid in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, we underwent analysis by bioinformatics. RESULTS: Forty-four colonies were sequenced, among which twenty-five colonies were immunoglobulin lambda light chain, six human DNA sequences from clone RP11-189K21, four human DNA sequences from clone RP11-507C10, two homo sapiens 12p BAC RPCI11-75L1, one homo sapiens BAC clone RP11-21M10, one homo sapiens ubiquitin ligase mind bomb (MIB), one homo sapiens genomic DNA, chromosome 11 clone: RP11-86708, one human DNA sequence from clone RP3-509I19, one homo sapiens small nuclear ribonucleoprotein polypeptide G, one homo sapiens UMP-CMP kinase (UMP-CMPK), and a new gene. CONCLUSION: Genes of HCTP4 interacting proteins in leukocytes are successfully cloned and the results bring some new clues for studying the biological functions of HCTP4 and associated proteins.