【摘要】 目的酵母双杂交筛选与TEB4(MARCH-Ⅵ)有相互作用的蛋白,构建该蛋白基因序列的重组载体。方法以人TEB4为诱饵质粒,利用酵母双杂交技术筛选人淋巴瘤cDNA文库。在SD/-4培养基上共获得150多个阳性克隆,将酵母菌扩增后抽提质粒,转化大肠杆菌,进行序列分析,并经过回复杂交验证,发现了一个与TEB4相互作用的蛋白:ATP synthase F0 subunit6(ATP-C6)。PCR法扩增ATP-C6,亚克隆到质粒pCMV-HA中,获得重组质粒pCMV-HA-ATP-C6,酶切和测序进行鉴定。结果从人淋巴瘤cDNA文库筛选到一个与TEB4有相互作用的蛋白ATP-C6,并成功构建重组质粒pCMV-HA-ATP-C6。结论应用酵母双杂交系统从人淋巴瘤cDNA文库中筛选并初步验证了与TEB4有相互作用的蛋白,为TEB4蛋白功能的进一步研究奠定了基础。更多还原

【Abstract】 【Objective】 To screen the interactive proteins of TEB4(MARCH-VI)and construct the vector.【Methods】Yeast two-hybrid technique was performed to screen a human lymphoma cDNA library with pGBKT7TEB4 as bait plasmid.About one hundred and fifty positive clones were obtained from SD/-4 medium plate.The plasmids from yeast were transformed into E.coli DH5α and extracted for sequencing.After repetitive hybrid confirmation,the putative protein that can interact with TEB4 was ATP synthase F0 subunit 6.This gene was amplified by PCR method and cloned into the pCMV-HA vector,harvesting a recombinant plasmid pCMV-HA-ATP-C6.【Results】Only one positive clone identified by yeast two-hybrid,a recombinant plasmid pCMV-HA-ATP-C6 was successful constructed.【Conclusion】Screen and initially identify the interactive protein of TEB4 is a foundation for the study on the protein of TEB4.更多还原