本文選題：人脂聯(lián)素去信號(hào)肽區(qū) + 原核表達(dá)�。� 參考：《河南師范大學(xué)》2011年碩士論文

【摘要】：近年來(lái),肥胖癥、Ⅱ型糖尿病、動(dòng)脈粥樣硬化發(fā)病率不斷升高,血漿中脂聯(lián)素(adiponectin,ADPN)的含量與這類疾病的發(fā)生呈負(fù)相關(guān),通過檢測(cè)人血漿中脂聯(lián)素的含量可反應(yīng)這類疾病的發(fā)生狀況。本研究通過基因工程獲得了人脂聯(lián)素去信號(hào)肽區(qū)域重組蛋白,并以此制備標(biāo)準(zhǔn)品,初步建立了人脂聯(lián)素ELISA定量檢測(cè)方法。 根據(jù)NCBI上公布的人脂聯(lián)素基因序列NM_004797.2,利用primer5.0設(shè)計(jì)一對(duì)引物。從人脂肪組織中提取總RNA,通過RT-PCR獲得678bp的人脂聯(lián)素去信號(hào)肽區(qū)的基因片段,將目的片段和載體pET-41a分別進(jìn)行雙酶切,瓊脂糖凝膠電泳,回收純化目的片段和載體并進(jìn)行連接,將連接產(chǎn)物轉(zhuǎn)入DH5α,涂布LB固體培養(yǎng)基,挑取單菌落,擴(kuò)大培養(yǎng)后用堿裂解法提取質(zhì)粒,將雙酶切鑒定為陽(yáng)性的重組質(zhì)粒命名為pET-41a/ΔSADPN。將該重組質(zhì)粒轉(zhuǎn)入E.coli BL21(DE)3中,用IPTG進(jìn)行誘導(dǎo)表達(dá),SDS-PAGE表明重組蛋白以可溶性形式表達(dá),并具有較好的免疫反應(yīng)性。 通過誘導(dǎo)表達(dá)條件的優(yōu)化,確立了最佳的誘導(dǎo)條件為:25℃,IPTG濃度0.03mmol/L,誘導(dǎo)時(shí)間8h。融合蛋白含有組氨酸標(biāo)簽,用鎳離子親和層析純化目的蛋白,得到的融合蛋白溶液,經(jīng)檢測(cè)其濃度為2.1 mg/mL,純化后的蛋白制備成標(biāo)準(zhǔn)品。 以抗人脂聯(lián)素單克隆抗體作為包被抗體,采用改良的過碘酸鈉法,對(duì)人脂聯(lián)素多克隆抗體進(jìn)行辣根過氧化物酶標(biāo)記,作為標(biāo)記抗體。棋盤滴定法確立人脂聯(lián)素單克隆抗體的最佳包被濃度為1.0μg/mL;酶標(biāo)抗體的最佳稀釋倍數(shù)為1:4000;以脂聯(lián)素去信號(hào)肽區(qū)重組蛋白為標(biāo)準(zhǔn)品繪制出的標(biāo)準(zhǔn)曲線,線性范圍為1ng～30ng;與同類試劑盒進(jìn)行臨床試驗(yàn)比較,其檢測(cè)結(jié)果通過t檢驗(yàn),P0.05,沒有顯著性差異。 本研究通過基因工程方法得到了免疫反應(yīng)性較好的重組人脂聯(lián)素去信號(hào)肽蛋白,制備了檢測(cè)脂聯(lián)素的標(biāo)準(zhǔn)品,初步建立人脂聯(lián)素雙抗體夾心檢測(cè)方法,為進(jìn)一步研制人脂聯(lián)素ELISA定量檢測(cè)試劑盒奠定基礎(chǔ)。
[Abstract]:In recent years, the incidence of obesity, type 2 diabetes and atherosclerosis has been increasing. The plasma adiponectin ADPNs are negatively correlated with the occurrence of these diseases. Adiponectin levels in human plasma can reflect the occurrence of these diseases. In this study, the recombinant protein of human adiponectin de-signalling peptide region was obtained by genetic engineering, and the standard product was prepared, and the quantitative detection method of human adiponectin ELISA was established. According to the human adiponectin gene sequence NM004797.2 published on NCBI, a pair of primers were designed by primer5.0. Total RNAs were extracted from human adipose tissue. The gene fragment of human adiponectin designalling peptide region of 678bp was obtained by RT-PCR. The target fragment and vector pET-41a were digested by double enzyme, agarose gel electrophoresis, and purified fragment and vector were recovered and ligated. The ligation product was transferred into DH5 偽, coated with LB solid medium, and a single colony was picked up. After expanded culture, the plasmid was extracted by alkaline lysis method. The recombinant plasmid identified by double enzyme digestion was named pET-41a/ 螖 SADPN. The recombinant plasmid was transferred into E.coli BL21(DE)3 and expressed by IPTG. SDS-PAGE showed that the recombinant protein was expressed in soluble form and had good immunoreactivity. Through the optimization of the induced expression conditions, the optimal induction conditions were established as follows: the concentration of IPTG at 25 鈩，

本文編號(hào)：1777695