抗HIV-1 gp41二硫键稳定单链抗体及其重组导向毒素的表达及活性测定

发布时间：2019-01-26 15:02

【摘要】： 对已有的抗HIV-1gp41单链抗体(41)基因进行定点突变获得抗HIV-1gp41二硫键稳定单链抗体(d41)基因,同时通过PCR方法获得金黄色葡萄球菌外毒素(SEA′)基因,并将SEA′基因插入d41基因上游构建成重组导向毒素(sd41)基因。将d41和sd41基因分别克隆入原核表达载体pET-28a(+)中,获得pET-d41和pET-sd41原核表达质粒。利用大肠杆菌表达系统,对d41和sd41基因进行了原核表达。表达的目的蛋白主要以包涵体形式存在。通过Ni-NTA亲和层析的方法对包涵体成功地进行了纯化,并利用尿素梯度透析法对纯化蛋白复性。通过与亲本抗体比较,表明抗HIV-1gp41二硫键稳定的单链抗体(d41)及以其为弹头的重组导向毒素(sd41)抗原亲和性略有下降,但稳定性有明显提高。该结果为进一步研究抗HIV制剂奠定了坚实的基础。
[Abstract]:The HIV-1gp41 disulfide stable single chain antibody (d41) gene was obtained by site-directed mutagenesis of the existing anti HIV-1gp41 single chain antibody (41) gene. Staphylococcus aureus exotoxin (SEA') gene was obtained by PCR method. SEA' gene was inserted into the upstream of d41 gene to construct recombinant directed toxin (sd41) gene. The d41 and sd41 genes were cloned into the prokaryotic expression vector pET-28a () and the prokaryotic expression plasmids pET-d41 and pET-sd41 were obtained. The prokaryotic expression of d41 and sd41 genes was carried out by using Escherichia coli expression system. The expressed target protein mainly exists in the form of inclusion body. Inclusion bodies were successfully purified by Ni-NTA affinity chromatography and renatured by urea gradient dialysis. Compared with parental antibody, the affinity of single chain antibody (d41) and recombined directed toxin (sd41) antigen against HIV-1gp41 disulfide bond was decreased slightly, but the stability was improved obviously. The results laid a solid foundation for the further study of anti-HIV preparations.
【学位授予单位】：长春理工大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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