口疮病毒VEGF基因的原核表达及亚细胞定位

发布时间：2018-04-04 07:51

  本文选题：口疮病毒　切入点：VEGF基因　出处：《中国兽医科学》2017年02期

【摘要】：为了对口疮病毒(ORFV)VEGF基因进行原核表达和亚细胞定位研究,PCR扩增VEGF基因,并将其分别克隆至原核表达载体p ET-32a(+)和真核表达载体p EGFP-C3中。将原核表达重组质粒转化至E.coli Rosseta感受态细胞中,经IPTG诱导表达目的蛋白。将表达产物纯化后免疫BALB/c小鼠,制备多克隆抗体血清,并利用琼脂扩散试验检测其效价。将真核表达重组质粒转染BHK21细胞,利用激光共聚焦观察VEGF蛋白在BHK21细胞中的亚细胞定位。结果表明:VEGF蛋白在大肠杆菌中获得了高效表达,主要以包涵体形式表达,蛋白分子质量大小约为33 ku。Western-blot结果表明,VEGF蛋白具有良好的反应原性,制备的多克隆抗体效价达1∶8。VEGF蛋白在转染BHK21细胞后主要在细胞质中表达,且能够与制备的多抗血清反应。本研究为进一步研究该蛋白的功能奠定了一定的基础。
[Abstract]:In order to investigate the prokaryotic expression and subcellular localization of VEGF gene of oral ulcera virus, VEGF gene was amplified and cloned into prokaryotic expression vector pET-32a () and eukaryotic expression vector p EGFP-C3, respectively.The prokaryotic expression plasmid was transformed into E.coli Rosseta competent cells, and the target protein was induced by IPTG.The BALB/c mice were immunized with the purified product, and the polyclonal antibody serum was prepared, and the titer was detected by Agar diffusion test.The eukaryotic expression plasmid was transfected into BHK21 cells and the subcellular localization of VEGF protein in BHK21 cells was observed by confocal laser.The results showed that the protein was highly expressed in E. coli, mainly in the form of inclusion body. The molecular weight of the protein was about 33 ku.Western-blot.The polyclonal antibody titer of the prepared 1:8.VEGF protein was mainly expressed in the cytoplasm of BHK21 cells after transfection and could react with the prepared polyantiserum.This study laid a foundation for further study of the function of the protein.
【作者单位】： 安徽农业大学动物科技学院;
【基金】：国家自然科学基金项目(31602063) 安徽省自然科学基金项目(1508085QC60) 安徽省科技重大专项(16030801106) 安徽农业大学稳定和引进人才科研资助项目(yj2015-16) 国家现代农业产业技术体系专项基金项目(CARS-39)
【分类号】：S852.65
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本文编号：1709041