BCG活化小鼠巨噬细胞膜蛋白NP-2的基因克

发布时间：2018-02-07 17:02

  本文关键词： 活化巨噬细胞 原核表达 包涵体 多克隆抗体　出处：《吉林大学》2010年硕士论文　论文类型：学位论文

【摘要】： 本实验根据已报道的BCG活化巨噬细胞表面膜蛋白具有抗肿瘤作用,我们从相关蛋白质数据库中筛选出具有磷脂酶活性的一个蛋白做为研究对象。该蛋白IPI序列号为00221418,我们将其命名为NP-2(novel protein-2)。通过生物信息学分析,得知该蛋白相关的膜外区信息。接着我们用BCG刺激小鼠,在获得小鼠腹腔活化的巨噬细胞后,应用RT-PCR扩增NP-2膜外区基因,将PCR扩增产物与原核表达载体pGEX-4T1相连,得到重组质粒pGEX-4T1-NP-2,并转化到Rosetta (DE3)工程菌中表达,经过不同时间,不同浓度IPTG诱导表达,确定表达最佳条件和表达形式,最佳条件为IPTG浓度1.0mmol/L、37℃诱导6小时。在最佳表达条件下大量诱导表达重组质粒pGEX-4T1-NP-2,经稀释复性,GS4B亲和层析纯化和蛋白浓缩后,获得蛋白浓度为0.50mg/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。本研究首次克隆了NP-2膜外区基因、并进行表达,得到了相关蛋白,并对其进行了生物信息学分析,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, BCG activated macrophage surface membrane protein has anti-tumor effect. We selected a protein with phospholipase activity from a database of related proteins. The IPI sequence number of the protein was 00221418, and we named it NP-2(novel protein-2, which was analyzed by bioinformatics. We obtained the information about the extracellular region of the protein. Then we stimulated the mouse with BCG. After we obtained the activated macrophages in the abdominal cavity of mice, we amplified the NP-2 extracellular region gene by RT-PCR, and linked the PCR amplification product to the prokaryotic expression vector pGEX-4T1. The recombinant plasmid pGEX-4T1-NP-2 was obtained and transformed into Rosetta DE3). After different time and different concentration of IPTG, the optimal expression conditions and expression forms were determined. The optimal conditions were as follows: the concentration of IPTG was 1.0 mmol / L at 37 鈩，

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