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朱雷, 胡燕, 侯俊, 沈宏辉, 杨健洋, 貌盼勇. HIV多抗原位点串联基因的表达及活性鉴定[J]. 中国公共卫生, 2005, 21(2): 176-177.
引用本文: 朱雷, 胡燕, 侯俊, 沈宏辉, 杨健洋, 貌盼勇. HIV多抗原位点串联基因的表达及活性鉴定[J]. 中国公共卫生, 2005, 21(2): 176-177.
ZHU Lei, HU Yang, HOU Jun, . Expression and preliminary activity identification of several linked antigen epitopes of envelop genes of HIV[J]. Chinese Journal of Public Health, 2005, 21(2): 176-177.
Citation: ZHU Lei, HU Yang, HOU Jun, . Expression and preliminary activity identification of several linked antigen epitopes of envelop genes of HIV[J]. Chinese Journal of Public Health, 2005, 21(2): 176-177.

HIV多抗原位点串联基因的表达及活性鉴定

Expression and preliminary activity identification of several linked antigen epitopes of envelop genes of HIV

  • 摘要:
      目的   在原核表达载体系统中对HIV-1/2型外壳蛋白多个抗原位点串联基因进行表达、纯化并鉴定其活性。
      方法   人工合成含HIVgp41的2个抗原位点、gp120的3个群的各3个抗原位点及gp36的2个抗原位点的串联基因, 克隆到原核表达载体pRSETB中, 构建重组表达质粒pRSETBenv, 异丙基βD硫代半乳糖苷(IPTG)诱导其在大肠埃希菌BL21(DE3)中高效表达, 利用固化金属离子配体亲和层析技术纯化表达蛋白, 利用逐渐降低尿素浓度透析法使目的蛋白复性。用免疫印迹和ELISA法分别对表达产物进行鉴定。
      结果   目的基因在BL21中有较高表达率, 纯化后表达蛋白经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDSPAGE)可见一条约32KD的蛋白带, 与设计分子量相符。免疫印迹、ELISA试验显示该表达蛋白可与HIV患者血清较好结合, 而与其它患者血清无交叉反应。
      结论   成功构建了由HIV外膜蛋白多个抗原位点串联基因片段的表达载体pRSETBenv, 并在原核细胞中高效表达, 表达蛋白经一步纯化后纯度较高, 并具有良好特异性和活性。

     

    Abstract:
      Objective   To express the sever al linked antig en epitopes of envelop genes of HIV1/2 types in the system of prokaryotic expression vector; to purify and to identify the expressing products.
      Methods   In order to construct the recombinant expressing plasmid pRSET B-env, the linked genes contained two antigen epitopes of gp 41, three different antigen epitopes of gp 120 and two antigen epitopes of gp 36 were inserted into pRSETB vector.After transforming into E.coli BL 21 (DE3) cells.The recombinant protein was expressed with induction of isopropy 1β-D-thiogalactopyranoside(IPTG), and was purified by immobilized metalion affinity chromatograph and was recoveried by dialysis in urea of slowby decreasing concentration.The immunoactivity was analyzed by westernblot and Enzyme-linked immunosorbent assay(ELISA).
      Results   An expected about 32KD expressing protein band could be seen with sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE). The aim genes revealed higherexpressive rate in E.coli BL 21 (DE3) cells.The results of westernblot and ELISA showed specific reactions with HIV patients' sera, and no cross-reaction with other patients.ser a using the expressing protein.
      Conclusion   The recombinant expressing plasmid pRSET B-env which contained the several linked antigen epitopes of envelop genes of HIVwas successfully constructed.After purification, the expressing protein possessed higher pureness, good specificity and activity.

     

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