中国HIV-1 B’亚型流行株gp120蛋白的表达与纯化-东南大学学报医学版

中国HIV-1 B’亚型流行株gp120蛋白的表达与纯化

单位：南京大学医学院公共健康医学中心, 江苏南京 210093

分类号：Q51;Q786

出版年·卷·期（页码）：2013·32·第六期（742-745）

摘要：

目的：表达纯化中国HIV-1 B’亚型流行株gp120蛋白。方法：将gp120基因克隆至真核表达载体pTriEx-3 hygro，构建真核表达质粒pTriEx-3-gp120，转染CHO-K1细胞，96 h后收取上清，利用金属螯合层析的方法纯化该重组蛋白，并通过SDS-PAGE以及Western blotting验证重组蛋白的表达。结果：酶切及测序结果表明真核质粒表达构建正确，表达的重组蛋白可以被His标签抗体以及HIV-1阳性血清所识别，并从细胞转染上清中成功纯化了该蛋白。结论：我们成功构建了一个可用于免疫检测、疫苗设计以及其它相关研究的HIV-1免疫原。

Objective: To express and character a recombinant HIV-1 gp120 (rgp120) derived from the env gene of a Chinese B' subtype isolate. Methods: The gp120 gene was subcloned into pTriEx-3 hygro vector to construct eukaryotic expression plasmid pTriEx-3-gp120, which was transfected into CHO-K1 cell line. The expressed rgp 120 was purified by metal chelate affinity chromatography and characterized by SDS-PAGE and Western blotting. Results: The eukaryotic expression plasmid pTriEx-3-gp120 was successfully constructed and confirmed by enzyme digestion and sequencing. The rgp120 was identified by anti-his tag monoclonal antibody and HIV-1 positive sera, and can be isolated from cell culture supernatant by a simple and fast purification procedure based on metal chelate affinity chromatography. Conclusion: The protein may serve as a useful tool for studies relating to HIV-1 vaccine development, HIV-specific antibody responses and other relative research purposes.

参考文献：

[1] WALKER B D,BURTON D R.Toward an AIDS vaccine[J].Science,2008,320(5877):760-764.
[2] HU S L,STAMATATOS L.Prospects of HIV Env modification as an approach to HIV vaccine design[J].Curr HIV Res,2007,5(6):507-513.
[3] 程林,宋红勇,吴喜林,等.人CCR5基因真核表达质粒的构建及其鉴定[J].东南大学学报:医学版,2012,31(1):40-43.
[4] PARREN P W,MOORE J P,BURTON D R,et al.The neutralizing antibody response to HIV-1:viral evasion and escape from humoral immunity[J].AIDS,1999,13(Suppl A):137-162.
[5] FROST S D,WRIN T,SMITH D M,et al.Neutralizing antibody responses drive the evolution of human immunodeficiency virus type 1 envelope during recent HIV infection[J].Proc Natl Acad Sci USA,2005,102(51):18514-18519.
[6] 吴喜林,刘忠,程林,等.gp120蛋白多肽抗体的设计、制备与鉴定[J].东南大学学报:医学版,2012,31(3):286-290.

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