人肿瘤抑素相关T21RGD肽的表达与纯化

Expression and Purification of Recombinant Human Tumstatin Relevant T21RGD Peptide

目的构建引入RGD序列人肿瘤抑素相关21肽（T21RGD）-内含肽-几丁质结合蛋白融合表达载体，实现目的肽的几丁质珠亲和层析一步纯化。方法人工设计合成T21RGD肽基因，经重组定向克隆插入到原核表达载体pTYB2，酶切和测序鉴定正确后转化人大肠杆菌BL21（DE3），IPTG诱导其融合表达，对表达蛋白进行几丁质珠亲和层析，DTF诱导柱上裂解以获得T21RGD肽。经Trcine—SDS—PAGE和反相高效液相色谱（RP—HPLC）对纯化产物进行鉴定。结果质粒酶切和测序结果显示，含T21RGD肽基因按正确方向和序列插入质粒，融合蛋白表达量达菌体蛋白总量的50％以上，Trcine—SDS—PAGE显示了T21RGD肽目的条带与预期相符，反相高效液相色谱测定峰纯度达97％。结论成功构建T21RGD肽融合表达载体，实现了融合蛋白在大肠杆菌中高效表达和T21RGD肽几丁质珠亲和层析一步纯化，为进一步研究T21RGD肽抗肿瘤活性和作用机制奠定基础。

Objective To construct expression plasmid （pTYB2-T2 1RGD ）for Human Tumstatin Relevant T21RGD Peptide-intein-chitin binding protein, and to purify T21RGD peptide by one-step affinity chromatography. Methods Synthesized double-stranded oligomeric nucleotides encoding T21RGD peptide was cloned into the plasmid of pTYB2. The fusion protein was yielded in E. coli B21 （DE3） and purified by chitin affinity chromatography. T21RGD peptide was analyzed by Trcine-SDS-PAGE and reversed phase highperformance liquid chromatography （RP-HPLC）. Results Plasmid of pTYB2-T2 1RGD was correct in size, orientation and sequence, and the expression of fusion protein surpassed 50% of total bacteria protein. The band of T21RGD peptide was showed by Trcine-SDS-PAGE, and the purity of T21RGD peptide detected by RP-HPLC was up to 97%. Conclusion The recombinant expression vector pTYB2-T21RGD was successfully constructed and the efficiency was expressed. T21RGD peptide was purified by one-step chitin affinity chromatography, which established the foundation for further study of its anti-tumor activity and mechanism.