【作者】 张昕；

【导师】 王缦；

【作者基本信息】 华东理工大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 运用酶联免疫吸附方法检测HIV抗体的诊断试剂,因其筛选通量大、成本低、操作方便,而仍在艾滋病的初步诊断中被广泛采用。本论文以1型HIV病毒的跨膜蛋白gp41为研究对象,致力于以现代基因工程的方法,利用大肠杆菌原核表达系统,制备出具有良好免疫反应性的HIV-1gp41重组抗原蛋白,初步评价其在HIV-1抗体诊断试剂盒中的应用价值。本论文选取了编码HIV-1gp41中重要抗原表位的基因序列,设计了合适的引物对,进行PCR扩增,将其通过BamH I、Xho I双酶切后与质粒载体E3连接,转化入克隆宿主菌DH5α,抽提得到的重组载体继续转化表达宿主菌BL21(DE3),经IPTG诱导表达出重组蛋白,再根据重组蛋白含有的His-Tag,使用镍离子亲和柱进行蛋白纯化,最后将纯化、复性后的重组蛋白标记HRP,通过双抗原夹心酶联免疫法检测各种标本,以评价该重组抗原的免疫反应性。实验证明,本论文成功构建了含有HIV-1gp41重要抗原表位序列的表达载体和能够表达重组抗原的表达宿主菌株,并获得了具有较高纯度的目的重组蛋白。该重组抗原蛋白有优良的免疫反应性,在与相应抗体结合时具备良好的灵敏性、特异性和稳定性,且将其运用在检测HIV-1抗体的酶免法诊断试剂盒中时,通过与数个商品化同类试剂盒的比较,体现出了一定的优势,证明其在HIV-1抗体诊断试剂中具有潜在的应用价值。更多还原

【Abstract】 Enzyme-Linked Immuno Sorbent Assay (ELISA) is still a mainstream clinical screening method for the detection of HIV (Human Immunodeficiency Virus) infection, due to its high testing throughput, low-cost and easy to operate. This study aims to express HIV-1transmembrane glycoprotein gp41, which has fine immunoreactivity, in prokaryotic system of E.coli.The study seleted DNA sequences coding major epitopes of HIV-1gp41as template, designed suitable primers pair, and then amplified target genes through PCR. The genes, after digested by BamH I and Xho I, inserted into plasmid vector E3. Then the joint product was transported into cloning host DH5a, from which the recombinant plasmid was extracted and then further transpoted into expressive host BL21(DE3). After being induced with IPTG, the recombinant protein was obtained. Due to the His-Tag the protein contained, it was purified with Nif-chelated affinity chromatopraphy. After that, the recombinant antigen was labeled with HRP, and antigenicity was analysed by detecting various specimens through double-antigen sandwich ELISA.In this study, HIV-1transmembrane glycoprotein gp41was successfully expressed in E.coli system. This recombinant antigen that contained major epitopes of HIV-1gp41was further used as a raw material, to develop HIV-ELISA kit, for the detection of HIV antibody in human blood. The antigenicity and the immunoreactivity of the recombinant protein of HIV-1gp41was analysed. The comprehansive evaluation of its sensitivity, specificity and stability was performed by comparing with several commercially available ELISA kits of good brand. The results showed a preferable performance of the kit that contained this recombinant antigen, this may partially lead to a commercial utilization of the antigen in potential HIV antibody detection kit.更多还原