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当前位置: 首页 > 文章 > HIV-1可溶性单链抗体b12-scFv的表达与活性分析 西北农林科技大学学报(自然科学版) 2014,42 (5) 205-210+216
Position: Home > Articles > Expression and characterization of soluble single-chain antibody b12-scFv against HIV-1 Journal of Northwest A & F University(Natural Science Edition) 2014,42 (5) 205-210+216

HIV-1可溶性单链抗体b12-scFv的表达与活性分析

作  者：
杨雄;刘秀侠;南昊;许晓东;陈红英
单  位：
西北农林科技大学生命科学学院
关键词：
HIV-1;单链抗体;b12;载体构建;亲和纯化;抗原结合活性
摘  要：
【目的】合成并克隆HIV-1单克隆抗体b12的单链抗体基因,在大肠杆菌中诱导表达,并分析表达产物的活性。【方法】以单抗b12的重链和轻链全基因为模板,分别扩增其可变区基因片段后,用重叠PCR的方法将二者拼接起来,形成scFv-VL-Linker-VH结构,并将其插入pET28a载体,构建原核表达载体pETb12-scFv。将pETb12-scFv转入大肠杆菌BL21StarTM(DE3),经IPTG诱导表达,对表达产物进行SDS-PAGE分析;用Ni-NTA金属螯合层析柱对表达产物进行分离纯化,用酶联免疫吸附试验(ELISA)、荧光显微镜和流式细胞仪(FCM)检测纯化蛋白与HIV-1gp120的结合活性。【结果】得到了b12的单链抗体基因b12-scFv,长度为768bp;SDS-PAGE电泳结果显示,b12-scFv表达量较高,蛋白质分子质量约为29ku,主要以可溶性蛋白的形式存在,但也有部分形成不溶的包涵体;纯化后蛋白的产量约为2mg/L;活性检测结果表明,b12-scFv对分泌表达和锚定在细胞膜表面的HIV-1gp120都具有良好的特异性结合活性。【结论】单链抗体b12-scFv可以用于HIV-1受体结合位点的表位检测。
译  名：
Expression and characterization of soluble single-chain antibody b12-scFv against HIV-1
作  者：
YANG Xiong;LIU Xiu-xia;NAN Hao;XU Xiao-dong;CHEN Hong-ying;College of Life Sciences,Northwest A&F University;
关键词：
HIV-1;;scFv;;b12;;vector construction;;affinity purification;;antigen binding activity
摘  要：
【Objective】The objective of this study was to synthesize,clone,express,purify,and characterize the scFv gene of antibody b12against HIV-1in Escherichia coli.【Method】The variable regions of the heavy and light chains of broadly neutralization antibody b12were amplified by polymerase chain reaction(PCR).The two regions were linked together as scFv-VL-Linker-VH by over-lap PCR and cloned into aprokaryotic expression vector pET28ato construct pET28a-b12-scFv,which was then transformed into E.coli BL21Star TM(DE3)cells for inducible expression by IPTG.The expression products were analyzed by SDS-PAGE and the protein was purified by affinity chromatography using Ni-NTA His Bind Resin. Then its binding activity with HIV-1 gp120 was examined by enzyme-linked immunosorbent assay(ELISA),immunofluorescence and flow cytometry(FCM).【Result】The soluble single-chain antibody b12-scFv(768bp)was obtained.SDS-PAGE showed that the expression of b12-scFv was high with a molecular weight of 29ku.The b12-scFv was mainly soluble with an insoluble part aggregated in inclusion body.The production of purified soluble b12-scFv was about 2mg/L.The b12-scFv was shown to have specific and good reactivity with both secreted and membrane anchored HIV-1gp120.【Conclusion】Theobtained single-chain antibody b12-scFv could be potentially applied in probing the receptor binding site on HIV-1gp120.

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