NGAL重组蛋白的表达与纯化及其促进结肠癌转移的研究  

Expression and Purification of the Recombinant NGAL Protein and Its Promotion of the Metastasis of the Human Colon Cancer

作　　者：刘桦[1,2] 杨晓龙[1] 唐丽[1] 胡开锋 刘明佳[1] 刘戟[1] 

机构地区：[1]四川大学华西基础医学与法医学院生物化学与分子生物学教研室,成都610041 [2]成都医学院生物医学系,成都610500

出　　处：《成都医学院学报》2015年第2期142-146,151,共6页Journal of Chengdu Medical College

基　　金：国家自然科学基金项目(No:J1103604)

摘　　要：目的研究NGAL重组蛋白促进人结肠癌细胞SW480转移的作用机制。方法通过基因工程方法构建重组表达载体pet32a-tev-NGAL,转化大肠杆菌(BL21-DE3)进行表达;采用亲和层析纯化,并用tev酶进行酶切获得NGAL重组蛋白;Transwell与基质胶实验检测NGAL对细胞迁移、侵袭能力的影响;Western blot检测NGAL对肿瘤转移相关蛋白表达的影响;ELISA检测不同肿瘤分期结肠癌患者血液中NGAL表达情况。结果成功构建pet32a-tev-NGAL原核表达载体,并通过体外表达与纯化获得NGAL重组蛋白;该蛋白促进SW480的迁移和侵袭;Western blot检测结果表明,NGAL激活EMT信号通路;ELISA结果表明,NGAL表达量与肿瘤恶化程度成正相关。结论 NGAL通过激活EMT信号通路促进结肠癌细胞SW480转移。Objective To investigate the biological functions and mechanisms of recombinant neutrophil gelatinase-associated lipocalin （NGAL） protein-induced colorectal cancer cells SW480 metastasis. Methods Pet32atev-NGAL recombinant expression vector was constructed and transferred into E. coli BL21. The fusion protein was purified by affinity chromatography and digested by the tev enzyme to obtain NGAL protein. The effect of NGAL protein on the invasive ability of metastasis was detected by Transwell and Matrigel experiments. NGAL expression was detected by Western blot on metastasis associated proteins. The expression levels of NGAL in the blood of colorectal carcinoma patients at different tumor stages were detected by ELISA. Results The construction of prokaryotic expression vector of pet32a-tev-NGAL was feasible. The NGAL recombinant protein can be expressed in E. coli BL21. NGAL promotes migration and invasion of SW480 cells. Western blot demonstrated that the NGAL could activate the EMT signaling pathway. ELISA results showed that there was a positive correlation between the expression level of NGAL and the degree of tumor malignancy. Conclusion NGAL promotes colorectal cancer SW480 cells metastasis by activating EMT signaling pathway.

关 键 词：NGAL重组蛋白 结肠癌细胞 肿瘤转移 

分 类 号：R735.35[医药卫生—肿瘤]