摘要
【目的】构建兔出血症病毒衣壳蛋白VP60基因的原核表达载体,进行原核表达并制备其蛋白抗体。【方法】以保存的VP60基因的质粒(pTeasy-VP60)为模版,用PCR方法获得VP60基因,并将其克隆到pET28a(+)载体上,构建重组表达载体pET28a(+)-VP60,酶切鉴定和测序验证后转入大肠杆菌BL21(DE3)中进行诱导表达,优化诱导表达时间和IPTG浓度;利用镍离子螯合层析纯化重组蛋白,并制备了高效价的抗VP60多克隆抗体。【结果】成功构建了兔出血症病毒衣壳蛋白VP60的原核表达质粒pET28a(+)-VP60,其在大肠杆菌中以包涵体形式高效表达,重组蛋白分子质量为60ku,制备的多克隆抗体具有较强的免疫结合活性。【结论】成功实现了VP60基因的原核表达,制备了重组蛋白VP60的多克隆抗体,为进一步的VP60转基因研究奠定了基础。
[Objective] The rabbit hemorrhagic disease virus（RHDV） capsid protein（VP60） gene was obtained by PCR,and cloned into prokaryotic expression vector pET28a（＋）. The identified construction was transformed into E. coli and over-expressed protein was purified by nickel chelating chromatography and polyclonal antiserum was raised against rabbit. [Method] The VP60 gene was amplified by PCR. The recombinant prokaryotic expression vector pET28a（＋）-VP60 was constructed by molecular technique, and then the recombinant wa~ transformed into E. coli BL21 （DE3） to induce protein expression with IPTG. The recombinant protein purified by nickel chelating chromatography was used as antigen to immunize rab- bit for preparation of polyclonal antibody. [Result] Prokaryotic expression vector pET28a（＋）-VP60 was successfully constructed, ~nd highly expressed in F.. coli BL21 （DE3） induced with IPTG, the molecular weight was 60 ku. Recombinant protein VP60 was purified by nickel chelating chromatography, and high ti- ter polyclonal antiserum was raised against rabbit. [Conclusion] Prokaryotic expression and polyclonal an- tibody preparation methods and techniques of VP60 gene were successfully established. The protein VP60polyclonal antibody has provided reliable tools for future study in the transgenic plant of VP60.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2012年第3期18-22,28,共6页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家转基因生物新品种培育科技重大专项(2008ZX08002)
国家自然科学基金项目(31071349)
关键词
兔出血症病毒衣壳蛋白
原核表达
多克隆抗体
rabbit hemorrhagic disease virus（RHDV） capsid protein
prokaryotic expression
polyclonalantibody
