人血管内皮生长因子受体-3胞外1-3区基因的表达

发布时间：2019-05-23 16:31

【摘要】：目的 1．血管内皮生长因子受体-3基因的原核表达载体构建 2．血管内皮生长因子受体-3基因的原核融合表达方法以本实验室构建的克隆质粒为模板，设计合适引物，在上游引物中加入酶切位点和起始密码，在下游引物中加入终止密码和酶切位点，，把上述VEGFR3扩增产物经切胶回收纯化后与pBV220质粒经双酶切纯化后的产物连接，把该质粒命名为pBV220-VEGFR3，转化大肠杆菌DH5α，筛选出阳性菌株，温控诱导表达，超声破菌，对包涵体进行洗涤纯化。以pBV220-VEGFR3为模板通过PCR方法构建C端含有HisTag的pBV220-VEGFR3his质粒并转化大肠杆菌DH5α，用Western-blotting鉴定，用Ni~(2+)-NTA树脂柱对表达产物进行纯化。结果构建了pBV220-VEGFR3质粒表达载体，SDS-PAGE电泳表明在大肠杆菌DH5α中成功表达出VEGFR3因子，同时构建了pBV220-VEGFR3融合表达载体，SDS-PAGE电泳和Western-blotting结果表明在大肠杆菌DH5α中成功表达出VEGFR3融合蛋白，用Ni~(2+)-NTA树脂柱纯化后获得VEGFR3融合蛋白纯品。结论应用基因工程技术，成功构建了pBV220-VEGFR3质粒表达载体，表达纯化出蛋白，为进一步的研究奠定了基础。
[Abstract]:Objective 1. Construction of prokaryotic expression vector of vascular endothelial growth factor receptor-3 gene. The prokaryotic fusion expression method was based on the cloned plasmid constructed in our laboratory as template. Appropriate primers were designed, enzyme digestion sites and initial codes were added to upstream primers, termination codes and enzyme digestion sites were added to downstream primers, and the VEGFR3 amplification products were recovered and purified by gel cutting and linked to the products purified by double enzyme digestion of pBV220 plasmid. The plasmid was named pBV220-VEGFR3, and transformed into E. coli DH5 伪. The positive strain was screened out, the expression was induced by temperature control, the inclusion body was washed and purified by ultrasonic broken bacteria. Using pBV220-VEGFR3 as template, the pBV220-VEGFR3his plasmid containing HisTag at C terminal was constructed by PCR and transformed into E. coli DH5 伪. The expressed product was identified by Western-blotting and purified by Ni~ (2)-NTA resin column. Results pBV220-VEGFR3 plasmid expression vector was constructed. SDS-PAGE electrophoresis showed that VEGFR3 factor was successfully expressed in E. coli DH5 伪, and pBV220-VEGFR3 fusion expression vector was constructed at the same time. The results of SDS-PAGE electrophoresis and Western-blotting showed that the VEGFR3 fusion protein was successfully expressed in E. coli DH5 伪. The pure VEGFR3 fusion protein was purified by Ni~ (2)-NTA resin column. Conclusion pBV220-VEGFR3 plasmid expression vector was successfully constructed by genetic engineering technique, and the protein was expressed and purified, which laid a foundation for further research.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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