HIV-1整合酶核心区可溶性表达及抑制剂筛选

中国生物工程杂志

2012, Vol. 32

Issue (03): 14-19

研究报告

HIV-1整合酶核心区可溶性表达及抑制剂筛选

何红秋, 贾渝跃

重庆市科学技术研究院重庆生物医药与器械研究中心重庆 401123

Soluble Expression and Inhibitor Screening of the Central Core Domain of HIV-1 Integrase

HE Hong-qiu, JIA Yu-yue

Chongqing Center for Biomedicines and Medical Equipment, Chongqing Academy of Science and Technology, Chongqing 401123, China

全文: PDF(545 KB) HTML

摘要：

为了实现HIV-1整合酶蛋白核心区 (central core domain of integrase, IN-CCD) 的可溶性表达,并建立以IN-CCD为靶点的抑制剂体外筛选方法,从包含F185K突变HIV-1 IN基因的质粒中经PCR扩增得到含有F185K突变的IN-CCD基因,克隆到pET28b载体上构建重组质粒pIN-CCD,转化pIN-CCD至E. coli BL21 (DE3)中经IPTG诱导、表达,Ni-亲和层析纯化,获得IN-CCD蛋白。修饰DNA底物,以链亲和素包被的磁珠为载体捕获DNA产物,结合酶联免疫吸附测定法(ELISA)检测IN-CCD的去整合活性,并筛选以IN-CCD为靶点的抑制剂。结果表明重组蛋白IN-CCD实现了高效可溶性表达,纯化后蛋白纯度达95%。建立的ELISA可以检测IN-CCD的去整合活性,且方法特异性和灵敏度好,可以实现高通量抑制剂筛选。从100个样品中筛选得到5个具有初步抑制IN-CCD去整合活性的样品。

关键词： HIV-1; 整合酶核心区; 可溶性表达; 纯化; 抑制剂筛选

Abstract:

In order to express the central core domain of HIV-1 integrase (IN-CCD) in a soluble form and establish a method for inhibitor screening targeting IN-CCD in vitro, the gene encoding the IN-CCD protein with F185K mutation was amplified from a plasmid containing the full length of IN gene. The gene product encoding IN-CCD protein with F185K mutation was introduced into the pET28b vector to construct a pIN-CCD recombinant plasmid, which was subsequently transformed into E. coli BL21 (DE3) and induced with IPTG. After cell lysis, the soluble IN-CCD in the supernatant was purified by Ni-affinity column chromatography. DNA substrate was modified, and streptavidin-coated magnetic beads were employed as a solid support to capture the DNA reaction product. Finally, an enzyme-linked immunosorbent assay (ELISA) was developed for the activity characterization of IN-CCD and subsequent inhibitor identification. The results showed that IN-CCD was highly expressed in soluble form; the purity of IN-CCD protein was about 95% after purification and dialysis. The ELISA established in this work was sensitive and specific for the detection of IN-CCD disintegration reaction, and could be used for the high-throughput screening of IN-CCD inhibitors. Five samples to be active IN-CCD disintegration inhibitors were screened out from 100 samples.

Key words: HIV-1 IN-CCD Soluble expression Purification Inhibitor screening

收稿日期: 2011-11-08 出版日期: 2012-03-25

ZTFLH:

Q786

基金资助:

重庆市科技攻关重点资助项目 (CSTC2010AB5101)

通讯作者: 贾渝跃,cqjiawa326@189.cn E-mail: cqjiawa326@189.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章

引用本文:

何红秋, 贾渝跃. HIV-1整合酶核心区可溶性表达及抑制剂筛选[J]. 中国生物工程杂志, 2012, 32(03): 14-19.

HE Hong-qiu, JIA Yu-yue. Soluble Expression and Inhibitor Screening of the Central Core Domain of HIV-1 Integrase. China Biotechnology, 2012, 32(03): 14-19.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I03/14

[1] 曹志亮,何玉仙. HIV-1中和抗体和基于抗体的疫苗设计. 中国生物工程杂志,2010, 30(12): 105-110. Cao Z L, He Y X. Neutralizing antibodies and antibody-based HIV-1 vaccine design. China Biotechnology, 2010, 30(12):105-110.

[2] Daar E S, Richaman D D. Confronting the emergence of drug-resistant HIV type 1: impact of antiretroviral therapy on individual and population resistance. AIDS Res Hum Retroviruses, 2005, 21(5): 343-357.

[3] Mathers B M, Degenhar L, Philips B, et al.Global epidemiology of injecting drug use and HIV among people who inject drugs: a systematic review. The Lancet, 2008, 372(9651): 1733-1745.

[4] 李芳琼,丁倩,詹金彪. HIV-1整合酶及其抑制剂的研究进展. 中国生物工程杂志,2008, 28(1): 80-86. Li F Q, Ding Q, Zhan J B. Progress in research on HIV-1 integrase and its inhibitors. China Biotechnology, 2008, 28(1): 80-86.

[5] Cherepanov P, Maertens G N, Hare S. Structrual insights into the retroviral DNA integration apparatus. Current Opinion on Structural Biology, 2011, 21(2): 249-256.

[6] Adamson C S, Freed E O. Novel approaches to inhibiting HIV-1 replication. Antiviral Research, 2010, 85(1): 119-141.

[7] Chiu T K, Davies D R. Structure and function of HIV-1 integrase. Current Topics in Medicinal Chemistry, 2004, 4(9): 965-977.

[8] Chow S A, Vincent K A, Ellison V, et al. Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus. Science, 1992, 255(5045): 723-726.

[9] Chow S A, Brown P O. Substrate features important for recognition and catalysis by human immunodeficiency virus type 1 integrase identified by using novel DNA substrates. Journal of Virology, 1994, 68(6): 3896-3907.

[10] Diamond T L, Bushman F D. Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay. Nucleic Acids Research, 2006, 34(21): 6116-6125.

[11] Pluymers W, De Clerq E, Debyser Z. HIV-1 integrase as a target for antiretroviral therapy: a review. Current Drug Targets Infectious Disorders, 2001, 1(12): 133-149.

[12] McNeely M, Hendrix J, Busschots K, et al. In vitro DNA tethering of HIV-1 integrase by the transcriptional coactivator LEDGF/p75. Journal of Molecular Biology, 2011, 410(5): 811-830.

[13] Grant P, Zolopa A. Integrase inhibitors: a clinical review of raltegravir and elvitegravir. Journal of HIV Therapy, 2008, 13(2): 36-39.

[14] He H Q, Ma X H, Liu B, et al. A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads. Acta Pharmacologica Sinica, 2008, 29(3): 397-404.

[15] He H Q, Liu B, Zhang X Y, et al. Development of a high-throughput assay for the HIV-1 integrase disintegration reaction. Science China Life Sciences, 2010, 53(2): 241-247.

[16] Inglese J, Johnson R L, Simeonov A, et al. High-throughput screening assays for the identification of chemical probes. Nature Chemical Biology, 2007, 3(8): 466-479.

[17] 程绍辉,马晓慧,何红秋,等. HIV-1整合酶蛋白的可溶性表达及功能研究. 中国生物工程杂志,2006, 26(1): 22-26 Cheng S H, Ma X H, He H Q, et al. Prokaryotic expression of integrase enzyme of HIV-1 and the function study of integrase protein. China Biotechnology, 2006, 26(1): 22-26.

[18] Jenkins T M, Hickman A B, Dyde F, et al. Catalytic domain of human immunodeficiency virus type 1 integrase: identification of a soluble mutant by systematic replacement of hydrophobic residues. Proceedings of the National Academy of Sciences of the United States of Ameirica, 1995, 92(13): 6057-6061.

[19] Wu J A, Attele A S, Zhang L, et al. Anti-HIV activity of medicinal herbs: usage and potential development. The American Journal of Chinese Medicine, 2001, 29(1): 68-81.

[20] Ahn H C, Lee S Y, Kim J W, et al. Binding aspects of baicalein to HIV-1 integrase. Molecular and Cells, 2001, 12(1): 127-130.

[21] Marchand C, Johnson A A, Karki R G, et al. Metal-dependent inhibition of HIV-1 integrase by beta-diketo acids and resistance of the soluble double-mutant (F185K/C280S). Molecular Pharmacology, 2003, 64(3): 600-609.

[1]	张玲,曹小丹,杨海旭,李文蕾. 连续流层析技术在亲和层析中的应用及生产放大评估[J]. 中国生物工程杂志, 2021, 41(6): 38-44.
[2]	邓通,周海胜,吴坚平,杨立荣. 基于分子伴侣策略提高NADPH依赖型醇脱氢酶的异源可溶性表达 ^*[J]. 中国生物工程杂志, 2020, 40(8): 24-32.
[3]	吕一凡,李更东,薛楠,吕国梁,时邵辉,王春生. **LbCpf1基因的原核表达、纯化与体外切割检测 ^***[J]. 中国生物工程杂志, 2020, 40(8): 41-48.
[4]	蒋丹丹,王云龙,李玉林,张怡青. 含RGD修饰的病毒样颗粒递送ICG靶向肿瘤的研究 ^*[J]. 中国生物工程杂志, 2020, 40(7): 22-29.
[5]	谢航航,白红妹,叶超,陈永俊,袁明翠,马雁冰. 易发生聚集的重组HBcAg病毒样颗粒的纯化^*[J]. 中国生物工程杂志, 2020, 40(5): 40-47.
[6]	位薇,常保根,王英,路福平,刘夫锋. Tau蛋白核心片段306~378的异源表达、纯化及聚集特性验证^*[J]. 中国生物工程杂志, 2020, 40(5): 22-29.
[7]	刘珍珍,田大勇. 狂犬病疫苗蔗糖密度梯度离心纯化工艺开发 ^*[J]. 中国生物工程杂志, 2020, 40(4): 25-33.
[8]	朱彤彤,杨磊,刘应保,孙文秀,张修国. 辣椒疫霉PcCRN20-C蛋白的表达纯化及结晶 ^*[J]. 中国生物工程杂志, 2020, 40(1-2): 116-123.
[9]	潘炳菊,张宛怡,申会涛,刘婷婷,李中媛,罗学刚,宋亚囝. 甘露寡糖分离纯化研究进展^*[J]. 中国生物工程杂志, 2020, 40(11): 90-95.
[10]	谢玉锋,韩雪梅,路福平. 副干酪乳杆菌β-葡糖苷酶的表达、纯化及酶学性质研究 ^*[J]. 中国生物工程杂志, 2019, 39(5): 72-79.
[11]	付大伟,孙莹莹,徐伟. 融合蛋白NusA-hRI的高效异源表达、纯化及活性分析[J]. 中国生物工程杂志, 2019, 39(3): 21-28.
[12]	景佳美,徐欣,王敏,彭如超,施一. 沙粒病毒聚合酶C端的表达纯化与结晶条件筛选 ^*[J]. 中国生物工程杂志, 2019, 39(12): 18-23.
[13]	朱梦露,王雪雨,刘鑫,路福平,孙登岳,秦慧民. 一种新型亮氨酸5-羟化酶NmLEH的异源表达、纯化及酶学性质分析 ^*[J]. 中国生物工程杂志, 2019, 39(12): 24-34.
[14]	许敏华,张晶晶,金小宝,李小波,王艳,马艳. 美洲大蠊内生菌几丁质酶基因的克隆、表达及其活性研究 ^*[J]. 中国生物工程杂志, 2019, 39(1): 31-37.
[15]	童超迪,吴坚平,杨立荣,徐刚. X射线衍射晶体法解析脱卤酶DehDIV-R结构的研究 ^*[J]. 中国生物工程杂志, 2018, 38(8): 19-25.

Viewed

Full text

Abstract

Cited

Shared

Discussed