| 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BL21 StarTM(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-1 gp120的结合活性。【结果】得到了b12的单链抗体基因b12-scFv，长度为768 bp；SDS-PAGE电泳结果显示，b12-scFv表达量较高，蛋白质分子质量约为29 ku，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2 mg/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-1 gp120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。 |
| 关键词: HIV-1 单链抗体 b12 载体构建 亲和纯化 抗原结合活性 |
| DOI： |
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| 基金项目:教育部新世纪优秀人才支持计划项目(NCET-09-0652) |
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| Expression and characterization of soluble single-chain antibody b12-scFv against HIV-1 |
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YANG Xiong,LIU Xiu-xia,NAN Hao,et al
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| Abstract: |
| 【Objective】The objective of this study was to synthesize,clone,express,purify,and characterize the scFv gene of antibody b12 against HIV-1 in Escherichia coli.【Method】The variable regions of the heavy and light chains of broadly neutralization antibody b12 were amplified by polymerase chain reaction (PCR).The two regions were linked together as scFv-VL-Linker-VH by over-lap PCR and cloned into a prokaryotic expression vector pET28a to construct pET28a-b12-scFv,which was then transformed into E.coli BL21 StarTM(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 (768 bp) was obtained.SDS-PAGE showed that the expression of b12-scFv was high with a molecular weight of 29 ku.The b12-scFv was mainly soluble with an insoluble part aggregated in inclusion body.The production of purified soluble b12-scFv was about 2 mg/L.The b12-scFv was shown to have specific and good reactivity with both secreted and membrane anchored HIV-1 gp120.【Conclusion】The obtained single-chain antibody b12-scFv could be potentially applied in probing the receptor binding site on HIV-1 gp120. |
| Key words: HIV-1 scFv b12 vector construction affinity purification antigen binding activity |
