摘要
目的构建SDF-1/54R的原核可溶表达载体,并考查其对CXCR7的抑制作用。方法将PCR扩增的SDF-1/54r基因插入带有GST标签的可溶表达载体pET-41a+,并转化BL21(DE3)菌株,IPTG诱导表达的融合蛋白GST-SDF-1/54R通过SDS-PAGE和Western blotting进行检测,并利用GST亲和层析纯化。纯化产物经肠激酶酶切和纯化后得到目的蛋白SDF-1/54R。用SDF-1/54R处理CXCR7高表达的MCF-7细胞,利用MTT和趋化实验评价其对乳腺癌细胞增殖和转移的影响。结果利用新构建的可溶表达系统成功得到了目的蛋白SDF-1/54R,MTT和趋化实验证实SDF-1/54R对乳腺癌细胞MCF-7的增殖和转移具有明显的抑制作用。结论重组载体表达的可溶蛋白SDF-1/54R是一种良好的CXCR7特异性拮抗剂,具有开发成抗肿瘤药物的潜在应用价值。
Objective To construct a soluble prokaryotic expression vector of the CXCR7-specific antagonist SDF-1/54R and evaluate its activity. Methods SDF-1/54r gene amplified by PCR was inserted into the soluble expression vector pET-41a＋engineered with GST fusion tag, and the recombinant vector was transformed into E. coli strain BL21 （DE3）. After IPTG induction of E. coli, the expressed recombinant protein was purified with GST affinity chromatography purification system and confirmed by SDS-PAGE and Western blotting assay. The target protein SDF-1/54R was obtained after digestion of the purified product with enterokinase. Breast cancer MCF-7 cells with high expression of CXCR7 was treated with SDF-1/54R and the cell proliferation and metastasis was evaluated with MTT and chemotaxis assays. Results The target protein SDF-1/54R obtained showed an obvious inhibitory effect on the proliferation and metastasis of MCF-7 cells as confirmed by MTT and chemotaxis assays. Conclusion SDF-1/54R is a good antagonist of CXCR7 and shows a potential value as an effective anti-cancer agent.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2014年第6期818-822,共5页
Journal of Southern Medical University
基金
国家自然科学基金(81101732)
科技部重大新药创制计划(2012ZX09103-301-016)
教育部高等学校博士学科点专项科研基金(20104433120013)
广州市珠江科技新星专项基金(2013J2200047)
温州市科技计划(Y20100001)~~
