摘要
目的:表达并纯化血管生成素(ANG)与增强型绿色荧光蛋白(EGFP)融合蛋白,以进一步探讨血管生成素的生物学功能。方法:PCR扩增人ANG基因,将其与EGFP基因融合后克隆到高表达载体pMFHT,构建了原核表达质粒pHIS鄄ANG鄄EGFP。该质粒转化大肠杆菌BL21(DE3),IPTG诱导表达,并用金属螯合亲和层析纯化目的蛋白。激光共聚焦显微镜和Westernblot鉴定融合蛋白的表达情况。结果:重组蛋白在大肠杆菌中得到高效表达,并从破菌上清中一步纯化目的蛋白,纯度可达95%以上。Westernblot分析表明表达产物具有良好的抗原性和特异性。诱导表达的菌体在激光共聚焦显微镜下可发射明亮的绿色荧光。结论:成功的表达并纯化了ANG鄄EGFP融合蛋白,利用EGFP的荧光示踪作用,该融合蛋白可用于血管生成素核转位和胞内共定位蛋白质研究。
Objective: To express and purify fusion protein from angiogenin(ANG) and enhanced green fluorescent protein (EGFP) to further study the function of angiogenin. Methods: Human angiogenin gene was amplified by PCR and fused with EGFP. The fusion gene was inserted into the prokaryotic expression vector pMFHT to construct recombinant expression plasmid pHIS-ANG-EGFP. The recombinant plasmid was transformed into E.coli BL21(DE3) to express fusion protein under IPTG induction. The target protein was purified by Ni-NTA affinity chromatography. The induced bacteria were observed by confocal laser scanning microscope, and the purified protein was tested by Western blotting. Results: The fusion protein was highly expressed in E.coli, and then the target protein was purified in one step to 95% of purity from bacterial supernatant by Ni-NTA affinity chromatography. Western blotting assay demonstrated the expressed ANG-EGFP had good antigenicity and high specificity. Moreover, the induced bacteria could emit bright green fluorescence by confocal laser scanning microscope. Conclusion: The successful expression and purification of ANG-EGFP fusion protein provides materials to clarify the process of nuclear translocation of angiogenin and to explore its mechanism of action in RNA transcription.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2005年第7期437-442,共6页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金资助项目(30171035)
