本文關(guān)鍵詞： 活化巨噬細(xì)胞 原核表達(dá) 包涵體 多克隆抗體　出處：《吉林大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 本實(shí)驗(yàn)根據(jù)已報(bào)道的BCG活化巨噬細(xì)胞表面膜蛋白具有抗腫瘤作用,我們從相關(guān)蛋白質(zhì)數(shù)據(jù)庫(kù)中篩選出具有磷脂酶活性的一個(gè)蛋白做為研究對(duì)象。該蛋白IPI序列號(hào)為00221418,我們將其命名為NP-2(novel protein-2)。通過(guò)生物信息學(xué)分析,得知該蛋白相關(guān)的膜外區(qū)信息。接著我們用BCG刺激小鼠,在獲得小鼠腹腔活化的巨噬細(xì)胞后,應(yīng)用RT-PCR擴(kuò)增NP-2膜外區(qū)基因,將PCR擴(kuò)增產(chǎn)物與原核表達(dá)載體pGEX-4T1相連,得到重組質(zhì)粒pGEX-4T1-NP-2,并轉(zhuǎn)化到Rosetta (DE3)工程菌中表達(dá),經(jīng)過(guò)不同時(shí)間,不同濃度IPTG誘導(dǎo)表達(dá),確定表達(dá)最佳條件和表達(dá)形式,最佳條件為IPTG濃度1.0mmol/L、37℃誘導(dǎo)6小時(shí)。在最佳表達(dá)條件下大量誘導(dǎo)表達(dá)重組質(zhì)粒pGEX-4T1-NP-2,經(jīng)稀釋復(fù)性,GS4B親和層析純化和蛋白濃縮后,獲得蛋白濃度為0.50mg/ml的重組蛋白,用純化的目的蛋白免疫家兔,制備多克隆抗體,經(jīng)免疫印記技術(shù)Western-bloting和ELISA檢測(cè),獲得效價(jià)為1:12800的特異性多克隆抗體。本研究首次克隆了NP-2膜外區(qū)基因、并進(jìn)行表達(dá),得到了相關(guān)蛋白,并對(duì)其進(jìn)行了生物信息學(xué)分析,為進(jìn)一步研究其生物學(xué)功能以及作用機(jī)制奠定了基礎(chǔ)。
[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，

本文編號(hào)：1494868