【摘要】： 對已有的抗HIV-1gp41單鏈抗體(41)基因進行定點突變獲得抗HIV-1gp41二硫鍵穩(wěn)定單鏈抗體(d41)基因,同時通過PCR方法獲得金黃色葡萄球菌外毒素(SEA′)基因,并將SEA′基因插入d41基因上游構建成重組導向毒素(sd41)基因。將d41和sd41基因分別克隆入原核表達載體pET-28a(+)中,獲得pET-d41和pET-sd41原核表達質粒。 利用大腸桿菌表達系統,對d41和sd41基因進行了原核表達。表達的目的蛋白主要以包涵體形式存在。通過Ni-NTA親和層析的方法對包涵體成功地進行了純化,并利用尿素梯度透析法對純化蛋白復性。 通過與親本抗體比較,表明抗HIV-1gp41二硫鍵穩(wěn)定的單鏈抗體(d41)及以其為彈頭的重組導向毒素(sd41)抗原親和性略有下降,但穩(wěn)定性有明顯提高。該結果為進一步研究抗HIV制劑奠定了堅實的基礎。
[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.
【學位授予單位】：長春理工大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

【參考文獻】

相關期刊論文 前3條

1 歐陽晶,王健偉,屈建國,洪濤;白喉毒素DT_(386)片段的表達純化及細胞毒性研究[J];中國生物工程雜志;2004年06期

2 龔奕,劉康達,周鉻,薛瓊,陳紹亮,湯釗猷;~(131)I-抗HBxAg人/鼠嵌合抗體在裸鼠人肝癌模型定位的初步研究[J];中華核醫(yī)學雜志;1997年02期

3 趙雪,張萌，李煥婁，，盧圣棟;抗艾滋病分子導向藥物CD_4V_1V_2－PE40的基因構建及表達[J];中國醫(yī)學科學院學報;1995年04期

本文編號：2415609