| 摘要: |
| 【目的】合成、克隆HIV单克降抗体VRC01的单链抗体基因，在大肠杆菌中诱导表达并进行纯化。【方法】根据GenBank上公布的VRC01抗体可变区的氨基酸序列合成引物，运用基因从头合成和重叠延伸PCR方法，克隆抗体VRC01的重链可变区（VH）和轻链可变区(VL)基因；用15肽Linker(Gly4Ser)3将VH和VL基因相连，得到单链抗体基因scFv-VH-Linker-VL和scFv-VL-Linker-VH，并分别将两者克隆到原核表达载体pET28a中,构建重组质粒后转入大肠杆菌BL21 (DE3+)中诱导表达，对表达产物进行SDS-PAGE和Western blot分析；将重组蛋白进行变性处理后，利用Ni-NTA金属螯合层析柱对表达产物进行初步纯化。【结果】得到了VRC01的2种单链抗体的基因scFv-VH-Linker-VL和scFv-VL-Linker-VH，其大小分别为770和768 bp。SDS-PAGE电泳结果显示，scFv-VH-Linker-VL在大肠杆菌中不表达或表达量极低；而scFv-VL-Linker-VH表达量较高，蛋白质分子质量约为29 ku，且主要以包涵体形式存在。Western blot检测结果证实，scFv-VL-Linker-VH可与His-Tag融合表达，并通过亲和层析法成功纯化获得了该单链抗体。【结论】成功构建、表达并纯化出了VRC01单链抗体，为进一步研究VRC01单链抗体的生物学特性奠定了基础。 |
| 关键词: 人类免疫缺陷病毒(HIV) 单链抗体 VRC01 重叠PCR 原核表达 纯化 |
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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）；西北农林科技大学人才专项 |
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| Cloning,prokaryotic expression and purification of single-chain variable fragment（scFv）of antibody VRC01 against HIV-1 |
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| Abstract: |
| 【Objective】The objective of this study was to synthesize,clone,express,and purify the scFv gene of antibody VRC01 to HIV-1 in E.coli.【Method】The primers were synthesized according to the amino acid sequences of the variable regions of antibody VRC01 in GenBank.The DNA coding for the variable regions of antibody VRC01 was obtained by de novo gene synthesis using overlap PCR.To construct scFv-VH-Linker-VL and scFv-VL-Linker-VH,the VH and VL were linked with (Gly4Ser)3 linker by gene splicing using overlap PCR.The two constructed scFvs were cloned into pET28a and transformed into E.coli strain BL21(DE3+) for inducible expression.The expression products were analyzed by SDS-PAGE and Western blot and scFv was purified from the inclusion bodies by Ni-NTA chromatography.【Result】The DNA constructs of scFv-VH-Linker-VL(770 bp) and scFv-VL-Linker-VH(768 bp) were obtained.The expression of scFv-VH-Linker-VL was not detectable using SDS-PAGE,whereas expression of scFv-VL-Linker-VH was high in the form of inclusion body,with a molecular weight of 29 ku.Western blot result showed that scFv-VL-Linker-VH was purified successfully.【Conclusion】The scFv VRC01 was constructed,expressed and purified successfully,and it benefited further studies on the biological function of scFv VRC01. |
| Key words: HIV single-chain variable fragment of an antibody VRC01 overlap PCR prokaryotic expression purification |
