基于增殖诱导配体新型抗肿瘤分子的构建及初步筛选

Construction and screening of anti-tumor molecule based on a proliferation-inducing ligand

目的构建针对APRIL的免疫抑制分子,并对其原核表达蛋白进行纯化及活性筛选。方法将可溶性增殖诱导配体(sAPRIL)cDNA进行突变并插入TELTh表位序列,突变体用pQE-80L/DH5α原核表达系统进行表达,表达蛋白经SDS-PAGE鉴定、Ni-NTA亲和层析纯化及Western印迹分析,最后检测纯化蛋白对Raji细胞增殖的影响。结果成功构建并表达了4种sAPRIL突变体:C端引入HELTh表位的sAPRIL突变体(Ⅰ)、N端引入HELTh表位的sAPRIL突变体(Ⅱ)、C端缺失45bp序列并引入HELTh表位的sAPRIL突变体(Ⅲ)、N端缺失45bp序列并引入HELTh表位的sAPRIL突变体(Ⅳ)。SDS-PAGE电泳、Western印迹分析均检测到相应的特异蛋白带。用Ni-NTA系统成功纯化表达的突变体蛋白。细胞试验表明,突变体Ⅰ、Ⅱ纯化蛋白具有明显的促细胞增殖活性,而突变体Ⅲ、Ⅳ纯化蛋白没有促细胞增殖活性。结论在4种sAPRIL突变体中初步筛选到不具有促细胞增殖活性的APRIL免疫抑制分子,为下一步确定该分子的免疫抑制功能奠定了物质基础。

Objective To construct, express, purify and screen immunosuppressive molecule against human soluble APRIL (a proliferation-inducing ligand). Methods The cDNA of soluble APRIL (sAPRIL) was mutated and TEL Th epitope was added. Mutants was expressed by pQE-80L/DH5α system, identified by SDS-PAGE and Western blotting, purified by Ni-NTA resin. Their activity on stimulating the proliferation of Raji cell was detected. Results Four sAPRIL mutants were constructed and expressed as follows: HEL Th epitope was added at C end (Ⅰ); HEL Th epitope was added at N end (Ⅱ); the last 45-bp DNA was deleted at C end and HEL Th epitope was added (Ⅲ); the first 45-bp DNA was deleted at N end and HEL Th epitope was added (Ⅳ). Specific protein bands according to mutant Ⅰ-Ⅳ were detected by SDS-PAGE and Western blotting. Mutant protein was purified by Ni-NTA successfully. Mutants Ⅰand Ⅱ promoted cell proliferation remarkably, and mutant Ⅲand Ⅳnot. Conclusion Four sAPRIL mutants was constructed and expressed successfully. Immunosuppressive molecule against sAPRIL that can not promote cell proliferation was screened out, and it laid a foundation for further study on their immunosuppressive function.