重组HIV-1包膜糖蛋白gp120和gp41的表达、纯化及初步应用

发布时间：2018-06-13 10:04

  本文选题：人类免疫缺陷病毒1型 + gp120　； 参考：《西北大学》2006年硕士论文

【摘要】：人类免疫缺陷病毒(HIV)是引起艾滋病的病原体,分为两型:HIV-1和HIV-2,主要为HIV-1。它和一般的病毒有明显的差别,首先是其基因组相对复杂,编码更多的蛋白质来调控病毒潜伏、激活、转录与复制等过程,其次是它在宿主个体内变异迅速。鉴于HIV-1包膜糖蛋白gp120和gp41是HIV-1的主要抗原成分,是检测HIV抗体诊断试剂盒的重要组成成分,也是制备抗gp120和gp41单克隆抗体必需的免疫原,应用广泛,意义十分重大。本研究利用分子生物学手段,将gp120和gp41的编码基因分别重组到毕赤酵母及大肠杆菌的表达载体中,并成功地进行了蛋白表达。 外膜糖蛋白gp120的编码基因的克隆和在毕赤酵母中的表达。以质粒pHXB2-gp160为模板进行PCR,扩增出gp120抗原的全长DNA(gp120L)和短片段DNA(gp120S),构建了酵母表达载体pPICZ α A-gp120L和pPICZ α A-gp120S。将重组质粒线性化后转化到毕赤酵母GS115细胞株,使gp120基因同源重组整合到毕赤酵母的染色体上,经过选择培养基、PCR鉴定、Zeocin抗性筛选后,将得到的阳性菌株进行甲醇诱导表达,利用SDS-PAGE和Western blot分析表达产物,结果表明gp120L基因没有得到表达,gp120S基因在酵母胞内有微量表达,表达产物为19 kD。 跨膜糖蛋白gp41短片段基因在大肠杆菌中的表达、纯化及初步应用。同样以pHXB2-gp160为模板进行PCR,扩增出gp41抗原短片段DNA,构建重组表达质粒pET32a-gp41S,转化大肠杆菌BL21(DE3)plys,用IPTG诱导表达,表达产物经过SDS-PAGE和Western blot分析证实35.5 kD的目的蛋白gp41S主要存在于包涵体中,凝胶扫描成像分析证实其表达量占菌体总蛋白的10%以上。包涵体经过洗涤、溶解和复性后,纯度可达90%以上,表明已获得高纯度的目的蛋白。用纯化的重组gp41蛋白作为包被抗原,应用间接ELISA法对4份HIV阳性血清中的gp41抗体进行检测,结果表明该纯化蛋白具有一定的抗原活性,能特异性地与HIV抗体反应。用该蛋白免疫小鼠,经过三次免疫后抗体效价可达
[Abstract]:Human immunodeficiency virus (HIV) is the cause of AIDS, divided into two types: HIV-1 and HIV-2, mainly HIV-1. It is different from other viruses. Firstly, its genome is relatively complex, and it encodes more proteins to regulate the processes of virus latency, activation, transcription and replication, and then it mutates rapidly in host individuals. Since HIV-1 envelope glycoprotein gp120 and gp41 are the main antigen components of HIV-1, they are important components of HIV antibody diagnostic kit, and they are also the necessary immunogen to prepare monoclonal antibodies against gp120 and gp41. They are widely used and of great significance. In this study, the coding genes of gp120 and gp41 were recombined into Pichia pastoris and Escherichia coli by molecular biology, and the protein was successfully expressed. Cloning and expression of Encoding Gene of Outer membrane glycoprotein gp120 in Pichia pastoris. Plasmid pHXB2-gp160 was used as template to amplify the full-length DNA-gp120L of gp120 antigen and its short fragment, pPICZ 伪 A-gp120L and pPICZ 伪 A-gp120S. The yeast expression vectors pPICZ 伪 A-gp120L and pPICZ 伪 A-gp120Swere constructed. The recombinant plasmid was linearized and transformed into Pichia pastoris GS115 cell line. The homologous recombinant of gp120 gene was integrated into the chromosome of Pichia pastoris. The expression products were analyzed by SDS-PAGE and Western blot. The results showed that gp120L gene was not expressed in yeast cells and the expression product was 19kD. Expression, purification and preliminary application of transmembrane glycoprotein gp41 short fragment gene in Escherichia coli. Using pHXB2-gp160 as template, the short fragment of gp41 antigen was amplified, and the recombinant expression plasmid pET32a-gp41Swas constructed. The recombinant plasmid pET32a-gp41Swas transformed into E. coli BL21DDE3plys. the expression product was induced by IPTG. The result of SDS-PAGE and Western blot analysis confirmed that the target protein gp41S of 35.5 KD mainly existed in inclusion body. Gel scanning imaging analysis confirmed that its expression was more than 10% of the total bacterial protein. After washing, dissolving and renaturation, the purity of inclusion body was over 90%, which indicated that the target protein with high purity had been obtained. The purified recombinant gp41 protein was used as the coating antigen to detect the gp41 antibody in 4 HIV-positive sera by indirect Elisa. The results showed that the purified protein had a certain antigenic activity and could react specifically with HIV antibody. The antibody titers of mice immunized with this protein were up to three times.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：Q789;R392

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