应用酵母双杂交系统筛选hALR的相互作用蛋白

【作者】 陈思强；

【导师】 姚汝华；

【作者基本信息】 华南理工大学 ， 发酵工程， 2003， 硕士

【摘要】 肝脏是人体内少有的能够进行快速再生的器官之一。除肝部分切除之外、多种肝炎病毒的感染以及各种不同的化学药物会导致肝脏的损伤,从而触发肝细胞的再生过程。因此,作为一种器官,组织再生的合适模型,肝脏损伤与再生的分子生物学机制一直是人们研究的热点之一,对开发治疗肝损伤药物具有十分重要的意义。目前已知的肝再生相关因子如肝细胞生长因子(HGF)、表皮生长因子(EGF)、胰岛素、转化生长因子α(TGF α)和胰岛素样生长因子Ⅰ、Ⅱ(IGF Ⅰ、Ⅱ)等均难于解释具有器官特异性的肝再生调控机制。一种新型的促肝细胞增殖因子—肝再生增强因子(augmenter of liver regeneration,ALR)是一种特殊的促肝细胞分裂原,在肝损伤修复过程中发挥重要作用。但到目前为止,有关它在肝再生过程中的分子生物学机制并没有阐明。克隆与ALR相互作用的蛋白质,将有助于阐明ALR的信号传导机制,进而为阐明肝脏损伤与再生的分子生物学机制提供理论基础。 本研究应用酵母双杂交系统从人肝组织cDNA文库中筛选与人肝再生增强因子(hALR)的相互作用蛋白。主要方法如下: 一、抽提胎儿肝组织的mRNA,逆转录聚合链式反应(RT-PCR)扩增到hALR基因读码框片断,将其定向重组入DNA-BD载体pGBKT7,并用限制性内切酶酶切和DNA测序分析来鉴定构建的“诱饵”质粒pGBKT7-hALR。 二、醋酸锂(LiAc)转化法将“诱饵”质粒转入酵母菌AH109,通过SDS-PAGE和westem blot方法检测重组菌AH109(pGBKT7-hALR)中是否有BD-hALR融合蛋白的表达。采用营养缺陷培养和β-半乳糖苷酶活性滤膜影印分析法检测该诱饵质粒能否自身激活报告基因表达。 三、将MATa型的重组菌AH109(pGBKT7-hALR)与预转化酵母菌Y187(MAT α型)的人肝组织cDNA文库接合。通过营养缺陷培养和X-α-Gal活性分析筛选与hALR相互作用的阳性侯选克隆。 四、lyticase法抽提阳性侯选克隆的酵母总DNA;转化E.coli Top10,扩增获得单一文库质粒;运用PCR和限制性内切酶酶切法将这些文库质粒分组;各组取一代表做回转实验排除假阳性;对确定的阳性克隆进行DNA测序,测序结果提交GenBank分析。 主要研究结果如下: 一、获得的378bp的DNA序列与文献报道的hALR基因序列一致,限制性酶切和DNA测序结果证实hALR以正确的方向插入pGBKT7的EcoRI/BamH酶切位点,且读码框架未发生改变。 二、重组菌AH109(pGBKT7-hALR)在选择性培养基SD/-Trp平板上培养4天,更多还原

【Abstract】 The liver is a unique organ, and first in line, the hepatocytes encounter the potential to proliferate during cell mass loss. The control of hepatic growth and regeneration has interested experimentalists for much of the 20th century. Augmenter of liver regeneration (ALR), one of the latest hepatotrophic factors, is known to be live-specific. ALR specifically stimulates proliferation of cultured primary hepatocytes in vitro and enhances liver regeneration after liver partial hapatectomization in vivo. But it displays no significant effect on the proliferation of non-hepatocytes or tumor cell lines derived from tissues other than liver. Because of the target-specific activity, ALR was different from various well known non-specific hepatic stimulators such as insulin, EGF, TGF- α , IGF-I and HGF, which can stimulate proliferation of wide variety of cell types. However, the signaling mechanisms of ALR has been unclear. According to those, we seek for the genes encoding ALR-associated proteins from the human liver cDNA library using yeast two-hybrid system.Main procedures and methods:1. A hALR cDNA fragment was obtained by extracting mRNA from human fetal liver, and RT-PCR. It was inserted into DNA-BD vector pGBKT7 in the proper orientation to create a vector pGBKT7-hALR. The constructed plasmid was identified by restriction endonucleases digesting and DNA sequencing.2. The pGBKT7-hALR was introduced into yeast AH 109 by LiAc transformation method. The recombinant protein BD-hALR expression in the transformed yeast AH109(pGBKT7-hALR) was identified by SDS-PAGE and western blot. Nutritional selection screening and colony-lift filter assay of β -gal were used to test the BD-hALR protein for transcriptional activation function before using it to screen a library.3. AH109(pGBKT7-hALR) was mated with the pretransformed human liver cDNA library and the putative positive clones were obtained by nutritional selection screening and X- α -Gal assay.4. The total DNA was isolated from the putative positive clones with lyticase method, and then the AD plasmids were rescued via transformation of E.coli. To sort colonies, the AD library inserts were amplified by PCR and the PCR products were characterized by digesting with restriction enzyme. Yeast mating was performed to eliminate falase positives. Finally, the AD library inserts of the true two-hybrid positives were sequenced by using GAL4 AD sequencing primer and compared with those in更多还原