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Laser scanning cytometry: principles and applications-an update.激光扫描细胞术：原理与应用——最新进展
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用于活细胞CD4结合测定的荧光HIV-1 gp120的工程设计与开发。

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays.

作者信息

Costantini Lindsey M, Irvin Susan C, Kennedy Steven C, Guo Feng, Goldstein Harris, Herold Betsy C, Snapp Erik L

机构信息

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA; Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

出版信息

Virology. 2015 Feb;476:240-248. doi: 10.1016/j.virol.2014.12.019. Epub 2014 Dec 30.

DOI:10.1016/j.virol.2014.12.019
PMID:25555152
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4323844/
Abstract

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs.

摘要

HIV-1包膜糖蛋白gp120在HIV病毒进入和感染的初始步骤中与宿主细胞受体CD4结合。这一过程是开发抑制性药物和中和抗体的一个有吸引力的靶点。为了研究gp120的结合和细胞内运输，我们构建了人源化gp120 JRFL HIV-1变体与绿色荧光蛋白（GFP）的荧光融合蛋白。Gp120-sfGFP用人类糖类进行糖基化，表达量高，并从培养的人类细胞中分泌出来。蛋白质动力学、质量控制和运输可以在活细胞中可视化。融合蛋白可以很容易地用不同的gp120变体或荧光蛋白进行修饰。最后，分泌的gp120-sfGFP能够进行灵敏且简便的结合测定，通过荧光成像或激光扫描细胞术在活细胞上定量筛选gp120-CD4结合的潜在抑制剂。这种适应性强的研究工具应有助于gp120细胞生物学的研究和新型抗HIV药物的开发。