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。
The present study was designed to study the prokaryotic expression and subcellular localization of Orf virus VEGF gene.The VEGF gene was amplified by PCR and cloned into the prokaryotic expression vector p ET-32 a（＋） and eukaryotic expression plasmid p EGFP-C3,respectively.After being transformed into E.coli Rosseta competent cells,expression of the VEGF protein was induced by IPTG.Then,the BALB/c mice were immunized with the purified VEGF proteins to prepare for polyclonal antibodies.The titer of antibodies was detected by agar diffusion test.BHK-21 cells were transfected with recombinant eukaryotic expression plasmid p EGFP-VEGF using Lipofectamine 2000.The subcellular localization of VEGF protein in BHK-21 cells was observed by using a laser scanning confocal microscope.The results showed that after being induced by IPTG,VEGF protein was highly expressed mainly in the form of inclusion bodies.The molecular weight of the expressed VEGF protein was about 33 ku.The results of Western-blot assay demonstrated that the expressed VEGF protein had good immunological activity.The titer of polyclonal antibody was up to 1 ∶ 8.After being transfected to BHK-21 cells,VEGF protein was mainly expressed and distributed in the cytoplasm of the BHK-21 cells.The expressed protein had the reactivity with the polyclonal antibodies.In conclusion,the present study successfully expressed VEGF protein,and analyzed the subcellular localization of VEGF protein.It will provide the biological materials and basis for further functional research of the VEGF protein.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2017年第2期179-184,共6页
Chinese Veterinary Science
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)
关键词
口疮病毒
VEGF基因
原核表达
亚细胞定位
orf virus
VEGF protein
prokaryotic expression
subcellular localization
