活化巨噬細(xì)胞膜蛋白ENPP-4基因的原核表達(dá)及PcAb的制備
發(fā)布時(shí)間：2018-10-23 16:21
【摘要】： 已有研究結(jié)果表明,通過(guò)BCG刺激活化的巨噬細(xì)胞表面膜蛋白具有腫瘤殺傷功能。我們課題組進(jìn)而對(duì)其相關(guān)活化蛋白進(jìn)行了檢測(cè),其中存在活化上調(diào)表達(dá)的有454種。前期工作中就曾選擇了數(shù)種具有跨膜結(jié)構(gòu)的未知功能活化蛋白,希望研究這些蛋白在巨噬細(xì)胞接觸殺傷機(jī)制中可能的作用。鑒于此,本文也選取了IPI編號(hào)為00225072的跨膜蛋白的膜外區(qū)作為研究對(duì)象,其命名為ENPP-4。首先通過(guò)生物信息網(wǎng)站查詢和預(yù)測(cè)了該蛋白相關(guān)結(jié)構(gòu)功能信息;繼而再使用BCG免疫C57BL/6純系小鼠的方法,提取了活化的腹腔巨噬細(xì)胞。通過(guò)RT-PCR進(jìn)行目的蛋白ENPP-4基因膜外區(qū)的克隆擴(kuò)增,并且成功構(gòu)建了pET28a-ENPP-4重組質(zhì)粒。經(jīng)測(cè)序正確后,將此重組質(zhì)粒轉(zhuǎn)化入Rosetta (DE3)工程菌實(shí)行表達(dá),通過(guò)不同時(shí)間、不同溫度以及不同濃度IPTG誘導(dǎo)表達(dá)優(yōu)化,最終確定IPTG濃度0.6ug/ml、溫度37℃誘導(dǎo)6小時(shí)最為該目的蛋白的最優(yōu)表達(dá)條件,并大量誘導(dǎo)表達(dá)得到的重組蛋白。通過(guò)稀釋復(fù)性、透析復(fù)性后獲得了濃度為0.443ug/ml,純度達(dá)90%以上的復(fù)性蛋白。然后應(yīng)用復(fù)性的目的蛋白免疫家兔,制備了兔多克隆抗體。使用Western-bloting和ELISA等方法的檢測(cè)結(jié)果,顯示多克隆抗體具備特異性;效價(jià)達(dá)到1:6400。應(yīng)用此制備的多克隆抗體對(duì)C57BL/6純系小鼠的肌肉、脾臟、腦、心臟、肝臟等12個(gè)臟器進(jìn)行了目的蛋白的免疫組織定位檢測(cè),發(fā)現(xiàn)該蛋白在脾臟、肌肉、胃和卵巢中呈現(xiàn)高表達(dá)。本研究初步定位明確了ENPP-4蛋白的相關(guān)分布趨勢(shì),為該蛋白的功能研究提供了實(shí)驗(yàn)基礎(chǔ)。
[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，
本文編號(hào)：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，
本文編號(hào)：2289803
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2289803.html
最近更新
教材專著
