富含T细胞表位的肿瘤成纤维细胞激活蛋白片段克隆表达及纯化

Cloning expression and purification of the polypeptide with abundant T cell epitopes from tumor fibroblast activation protein

目的：表达并纯化富含T细胞表位的肿瘤成纤维细胞激活蛋白（fibroblas tactivation protein，FAP）片段。方法：利用T细胞表位预测软件及酶切位点预测软件分析FAP全长，选取FAP蛋白内富含T细胞表位又缺少酶切位点的多肽AA264～AA449（NP-032012．1）作为目的表达片段。该多肽所对应的核酸片段为mRNA798～1418（GenBank：BC019190．1）。利用PCR技术从含有FAP基因全长的质粒pCDNA3．1-FAP中扩增目的基因mRNA798～1418，并将该基因与原核表达载体pET-28a（＋）连接，在BL21工程菌中实现重组蛋白His6-FAP的表达，并利用His标签进行纯化。结果：成功构建了富含T细胞表位的FAP原核表达质粒pET-28a（＋）-FAP对该质粒进行BamHl／Xhol双酶切鉴定，鉴定结果与预期完全一致；实现了对该质粒编码的融合蛋白His6-FAP2的表达，表达后的融合蛋白主要以包涵体的形式存在；利用His标签纯化了该融合蛋白，纯化后灰度分析其纯度达95％。结论：成功表达并纯化了富舍细胞表位的FAP片段，为下一步以FAP为靶标的肿瘤免疫治疗策略提供了实验基础。

OBJECTIVE：To express and purify the tumor fibroblast activation protein （FAP） fragment enriched T cell epitopes. METHODS： Amino acid sequence of FAP was analysed by SYFPEITHI software combined with PAProC software,and then target fragment AA264-AA449 （NP_032012.1） with abundant T cell epitopes and lack of cleavage sites of protease were selected. The corresponding target gene for the polypeptide was mRNA798-1418 （GenBank： BC019190.1）. Gene fragment mRNA798-1418 was amplified from pCDNA3.1-FAP by PCR and cloned into pET 28a（＋ ） vector,the recombinant protein His6-FAP264-449 was expressed in BL21 engineering bacteria and then purified by His tag. RESULTS： The prokaryotic expression plasmid of FAP with abundant T cell epitopes was successfully constructed,and the results identified by double digestion with BamH1/Xhol corresponded exactly well as expected. The fusion protein His6-FAP264-449 was expressed and mainly existed in the form of inclusion body. Then the fusion protein was purified,its purity reached 95% through the gray-scale analysis. CONCLUSION： The FAP fragment enriched T cell epitopes was ex- pressed and purified successfully,that provided a basis for tumor immunotherapy targeting FAP.