摘要
目的:构建小鼠成纤维细胞激活蛋白α(FAPα)二肽基肽酶催化核心序列(FAPαc)的高效原核表达载体,进行该序列的原核表达、纯化和复性,为下一步高效抗体制作和以FAPα为靶标的抗肿瘤疫苗研发提供实验基础。方法:用PCR技术,从质粒pSecTag2/Hy-groB-FAPα中扩增出编码FAPα二肽基肽酶催化核心区域的基因片段,与原核表达载体pET28α(+)连接,在BL21(DE3)工程菌中实现重组蛋白His6-FAPαc的表达,并进行纯化和复性。结果:成功构建了小鼠来源的FAPα二肽基肽酶催化核心区域的原核表达质粒,通过对转化该质粒后的感受态细菌进行菌液PCR鉴定以及对该质粒进行BamHI和XhoI双酶切鉴定,鉴定结果与预期结果完全一致;实现了对该质粒编码的融合蛋白His6-FAPαc的表达,表达后的融合蛋白主要以包涵体的形式存在,表达产量占菌体总蛋白>70%;纯化了该融合蛋白,纯化后的蛋白纯度通过灰度分析>99%;对该融合蛋白进行了复性,复性后该融合蛋白由不溶的包涵体状态变为可溶的水溶状态。结论:成功表达、纯化和复性了FAPα二肽酶催化核心区域FAPαc,为深入开展FAPα的研究奠定基础。
OBJECTIVE： To construct a prokaryotic expression plasmid of the main catalytic domain of dipeptidyl peptidase of murine fibroblast activation protein a （FAPαc） and obtain the target fusion protein in order to provide basis for production of antibodies and anti-tumor vaccine targeting FAPα. METHODS： The gene fragment encoding catalytic domain of dipeptidyl peptidase of FAPα was amplified from plasmid pSecTag2/HygroB-FAPα by PCR and cloned into pET28α（＋） vector, the recombinant protein His6-FAPαc was expressed in BL21（DE3） engineering bacteria, then the target fusion protein was purified and renatured. RESULTS：The prokaryotic expression plasmid in the catalytic domain of dipeptidyl peptidase of FAPα was successfully constructed, and the results identified by PCR and digested by BamH Ⅰ and Xho Ⅰ corresponded exactly well as expected. The fusion protein His6-FAPαc was expressed and mainly existed in the form of inclusion body. His6- FAPαc took more than 70% of the bacterial protein. The fusion protein was purified, the purity reached more than 990% through the gray-scale analysis. The fusion protein was renatured, and after being renatured, the fusion protein came into a state of the water soluble from the insoluble inclusion bodies. CONCLUSION： The catalytic domain of dipeptidy Ⅰ peptidase of FAPα is expressed, purified and renatured successfully, establishing a foundation of further research of FAPα.
出处
《中华肿瘤防治杂志》
CAS
2009年第16期1206-1209,共4页
Chinese Journal of Cancer Prevention and Treatment
基金
国家自然科学基金(30873032)
国家自然科学基金(30672510)
广东省科技计划项目(2007B030704007)
关键词
成纤维细胞激活蛋白α
原核表达
纯化
复性
fibroblast activation protein α prokaryotie expression
purification
renaturation
