High-level expression of HIV-1 gp41 gene and purification of its product

BI Lan; LEI Xiao-Jun; CHEN Wei; YAN Jia-Xin; YU Mo-Song; SHU Jia-Hong

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August 1998

BI Lan, LEI Xiao-Jun, CHEN Wei, YAN Jia-Xin, YU Mo-Song and SHU Jia-Hong. High-level expression of HIV-1 gp41 gene and purification of its product[J]. Virologica Sinica, 1998, 13(4).

Citation: BI Lan, LEI Xiao-Jun, CHEN Wei, YAN Jia-Xin, YU Mo-Song, SHU Jia-Hong. High-level expression of HIV-1 gp41 gene and purification of its product .VIROLOGICA SINICA, 1998, 13(4) : 344.

HIV-1 gp41基因的高效表达及表达产物的纯化

1.
卫生部武汉生物制品研究所基因工程室

摘要

为了获得高效表达的人类免疫缺陷病毒（ＨＩＶ１）ｇｐ４１蛋白，从而为ＨＩＶ１基因工程诊断抗原的国产化打下基础，用ＰＣＲ的方法从ＨＩＶ１全基因序列中扩增出编码ｇｐ４１Ｎ端的６９０ｂｐ片段。经酶切后，克隆到ｐＥＴ２８ａ载体中，再将重组质粒转化到表达宿主菌ＢＬ２１（ＤＥ３）中，经ＩＰＴＧ诱导，高效表达出ｇｐ４１蛋白。间接ＥＬＩＳＡ、Ｗｅｓｔｅｒｎｂｌｏｔ、ＳＤＳＰＡＧＥ电泳证实，该表达产物具有良好的抗原性和特异性，且表达量约占总菌体蛋白的４５％。重组蛋白经金属鏊合纯化，纯度达９９％。
- 人免疫缺陷病毒-Ⅰ gp41基因 pET载体大肠杆菌BL21（DE3）高效表达

High-level expression of HIV-1 gp41 gene and purification of its product

1.
Department of Engineering．Wuhan lnstitute of Biological ProductsMinistry of Public Health，Wuhan 430O6O

Abstract

In order to get high level expression of HIV 1 gp41 gene, a fragment (690 bp) encoding N terminal of gp41 from all sequence of HIV 1 was amplified by PCR. After digesting by restriction enzyme EcoRI/Sal I, the fragment was cloned into pET28a plasmid which was digested by the same restriction enzyme, then recombinant plasmid was transformed into the E.coli BL21 (DE3). After inducing by IPTG, the bacteria produced the protein. Then the protein was purified through chelating Sepharose. Using indirect ELISA, SDS PAGE and Western blot test, it was found that the protein had good antigenicity, good specificity and high purification (99%), and its yield was about 45% of the total bacteria protein.
- HIV 1 gp41 gene pET vector E.coli BL21（DE3） High level expression

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

High-level expression of HIV-1 gp41 gene and purification of its product

1. Department of Engineering．Wuhan lnstitute of Biological ProductsMinistry of Public Health，Wuhan 430O6O

Abstract: In order to get high level expression of HIV 1 gp41 gene, a fragment (690 bp) encoding N terminal of gp41 from all sequence of HIV 1 was amplified by PCR. After digesting by restriction enzyme EcoRI/Sal I, the fragment was cloned into pET28a plasmid which was digested by the same restriction enzyme, then recombinant plasmid was transformed into the E.coli BL21 (DE3). After inducing by IPTG, the bacteria produced the protein. Then the protein was purified through chelating Sepharose. Using indirect ELISA, SDS PAGE and Western blot test, it was found that the protein had good antigenicity, good specificity and high purification (99%), and its yield was about 45% of the total bacteria protein.