本文選題：活化巨噬細(xì)胞 + 原核表達(dá)　； 參考：《吉林大學(xué)》2009年碩士論文

【摘要】： 本研究根據(jù)已報(bào)道的活化巨噬細(xì)胞表面膜蛋白的膜外區(qū)具有抗腫瘤作用，我們對(duì)相關(guān)蛋白數(shù)據(jù)庫進(jìn)行篩選，選取IPI序列號(hào)為00226142的蛋白為目的蛋白，并將其命名為AMMP-2（active macrophage membrane protein-2）。通過NCBI數(shù)據(jù)庫查詢其基因序列，進(jìn)行生物信息學(xué)分析，得到相關(guān)的膜外區(qū)信息。然后用BCG刺激小鼠，在獲得鼠腹腔活化的巨噬細(xì)胞基礎(chǔ)上，采用RT-PCR的方法擴(kuò)增AMMP-2膜外區(qū)基因，將獲得的PCR產(chǎn)物插入原核表達(dá)載體pET28a、pGEX4T1中，得到重組質(zhì)粒（pET28a-AMMP-2、pGEX4T1-AMMP-2），并將重組質(zhì)粒轉(zhuǎn)化到Rosetta (DE3)工程菌中進(jìn)行表達(dá)，經(jīng)過不同時(shí)間、溫度以及不同濃度IPTG誘導(dǎo)表達(dá)，確定最佳表達(dá)條件和表達(dá)形式，結(jié)果顯示最佳條件為：IPTG濃度0.8mmol/L、37℃誘導(dǎo)6小時(shí)，且均以包涵體形式表達(dá)。在最佳條件下重組質(zhì)粒pET28a-AMMP-2全菌表達(dá)量為31%大于重組質(zhì)粒pGEX4T1-AMMP-2的全菌表達(dá)量的24%。在最適條件下大量誘導(dǎo)表達(dá)重組質(zhì)粒pET28a-AMMP-2，獲得蛋白，經(jīng)稀釋復(fù)性、透析復(fù)性和CM纖維素陽離子交換純化，獲得純化后濃度為0.52mg/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，獲得效價(jià)為1：12800的特異性多克隆抗體。最后進(jìn)行了蛋白的免疫組織定位檢測，我們發(fā)現(xiàn)該蛋白在C57BL/6純系小鼠的肌肉、肝臟、腦組織中呈高表達(dá)。本研究首次進(jìn)行AMMP-2基因的克隆、表達(dá)及蛋白提純，并進(jìn)行了組織學(xué)定位分析，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, 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

本文編號(hào)：1753620