活化巨噬细胞膜蛋白AMMP-2基因的克

发布时间：2018-04-15 10:02

  本文选题：活化巨噬细胞 + 原核表达　； 参考：《吉林大学》2009年硕士论文

【摘要】： 本研究根据已报道的活化巨噬细胞表面膜蛋白的膜外区具有抗肿瘤作用，我们对相关蛋白数据库进行筛选，选取IPI序列号为00226142的蛋白为目的蛋白，并将其命名为AMMP-2（active macrophage membrane protein-2）。通过NCBI数据库查询其基因序列，进行生物信息学分析，得到相关的膜外区信息。然后用BCG刺激小鼠，在获得鼠腹腔活化的巨噬细胞基础上，采用RT-PCR的方法扩增AMMP-2膜外区基因，将获得的PCR产物插入原核表达载体pET28a、pGEX4T1中，得到重组质粒（pET28a-AMMP-2、pGEX4T1-AMMP-2），并将重组质粒转化到Rosetta (DE3)工程菌中进行表达，经过不同时间、温度以及不同浓度IPTG诱导表达，确定最佳表达条件和表达形式，结果显示最佳条件为：IPTG浓度0.8mmol/L、37℃诱导6小时，且均以包涵体形式表达。在最佳条件下重组质粒pET28a-AMMP-2全菌表达量为31%大于重组质粒pGEX4T1-AMMP-2的全菌表达量的24%。在最适条件下大量诱导表达重组质粒pET28a-AMMP-2，获得蛋白，经稀释复性、透析复性和CM纤维素阳离子交换纯化，获得纯化后浓度为0.52mg/ml的重组蛋白，用纯化的目的蛋白免疫家兔，，制备多克隆抗体，经免疫印记技术Western-bloting和ELISA检测，获得效价为1：12800的特异性多克隆抗体。最后进行了蛋白的免疫组织定位检测，我们发现该蛋白在C57BL/6纯系小鼠的肌肉、肝脏、脑组织中呈高表达。本研究首次进行AMMP-2基因的克隆、表达及蛋白提纯，并进行了组织学定位分析，为进一步研究其生物学功能以及作用机制奠定了基础。
[Abstract]:In this study, the extracellular region of activated macrophage surface membrane protein had anti-tumor effect. We screened the related protein database and selected the protein with IPI sequence number of 00226142 as the target protein, and named it AMMP-2(active macrophage membrane protein-2.The gene sequence was queried by NCBI database and bioinformatics analysis was carried out to obtain the information about the extracellular region.Then BCG was used to stimulate mice. On the basis of murine peritoneal activated macrophages, the extracellular region of AMMP-2 gene was amplified by RT-PCR, and the obtained PCR product was inserted into the prokaryotic expression vector pET28aNpGEX4T1.The recombinant plasmid pET28a-AMMP-2pGEX4T1-AMMP-2 was obtained, and the recombinant plasmid was transformed into Rosetta DE3 to express the recombinant plasmid. After different time, temperature and concentration of IPTG, the optimal expression conditions and expression forms were determined.The results showed that the optimal conditions were as follows: 1. The concentration of IPTG was 0.8 mmol / L for 6 hours at 37 鈩

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