活化巨噬细胞膜蛋白ENPP-4基因的原核表达及PcAb的制备
发布时间：2018-10-23 16:21
【摘要】： 已有研究结果表明,通过BCG刺激活化的巨噬细胞表面膜蛋白具有肿瘤杀伤功能。我们课题组进而对其相关活化蛋白进行了检测,其中存在活化上调表达的有454种。前期工作中就曾选择了数种具有跨膜结构的未知功能活化蛋白,希望研究这些蛋白在巨噬细胞接触杀伤机制中可能的作用。鉴于此,本文也选取了IPI编号为00225072的跨膜蛋白的膜外区作为研究对象,其命名为ENPP-4。首先通过生物信息网站查询和预测了该蛋白相关结构功能信息;继而再使用BCG免疫C57BL/6纯系小鼠的方法,提取了活化的腹腔巨噬细胞。通过RT-PCR进行目的蛋白ENPP-4基因膜外区的克隆扩增,并且成功构建了pET28a-ENPP-4重组质粒。经测序正确后,将此重组质粒转化入Rosetta (DE3)工程菌实行表达,通过不同时间、不同温度以及不同浓度IPTG诱导表达优化,最终确定IPTG浓度0.6ug/ml、温度37℃诱导6小时最为该目的蛋白的最优表达条件,并大量诱导表达得到的重组蛋白。通过稀释复性、透析复性后获得了浓度为0.443ug/ml,纯度达90%以上的复性蛋白。然后应用复性的目的蛋白免疫家兔,制备了兔多克隆抗体。使用Western-bloting和ELISA等方法的检测结果,显示多克隆抗体具备特异性;效价达到1:6400。应用此制备的多克隆抗体对C57BL/6纯系小鼠的肌肉、脾脏、脑、心脏、肝脏等12个脏器进行了目的蛋白的免疫组织定位检测,发现该蛋白在脾脏、肌肉、胃和卵巢中呈现高表达。本研究初步定位明确了ENPP-4蛋白的相关分布趋势,为该蛋白的功能研究提供了实验基础。
[Abstract]:It has been shown that macrophage surface membrane protein activated by BCG has tumor killing function. Our group then detected its related activation proteins, among which there were 454 kinds of activation-up-regulated proteins. Several unknown functional activating proteins with transmembrane structure have been selected in previous work to study the possible role of these proteins in the mechanism of macrophage contact killing. In view of this, the extracellular region of transmembrane protein with IPI number 00225072 was chosen as the research object, named ENPP-4.. The information about the structure and function of the protein was first queried and predicted by the bioinformatics website, and then the activated peritoneal macrophages were extracted by immunizing C57BL/6 mice with BCG. The extracellular region of the target protein ENPP-4 gene was cloned and amplified by RT-PCR, and the recombinant plasmid of pET28a-ENPP-4 was constructed successfully. After sequencing, the recombinant plasmid was transformed into Rosetta (DE3) engineering bacteria for expression, and the expression was optimized by different time, different temperature and different concentration of IPTG. The optimal expression conditions of the target protein were determined as follows: the concentration of IPTG was 0.6ugml, the temperature was 37 鈩，
本文编号：2289803
[Abstract]:It has been shown that macrophage surface membrane protein activated by BCG has tumor killing function. Our group then detected its related activation proteins, among which there were 454 kinds of activation-up-regulated proteins. Several unknown functional activating proteins with transmembrane structure have been selected in previous work to study the possible role of these proteins in the mechanism of macrophage contact killing. In view of this, the extracellular region of transmembrane protein with IPI number 00225072 was chosen as the research object, named ENPP-4.. The information about the structure and function of the protein was first queried and predicted by the bioinformatics website, and then the activated peritoneal macrophages were extracted by immunizing C57BL/6 mice with BCG. The extracellular region of the target protein ENPP-4 gene was cloned and amplified by RT-PCR, and the recombinant plasmid of pET28a-ENPP-4 was constructed successfully. After sequencing, the recombinant plasmid was transformed into Rosetta (DE3) engineering bacteria for expression, and the expression was optimized by different time, different temperature and different concentration of IPTG. The optimal expression conditions of the target protein were determined as follows: the concentration of IPTG was 0.6ugml, the temperature was 37 鈩，
本文编号：2289803
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