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目的克隆人增殖诱导配体(APRIL)基因,分析APRIL蛋白的生物学活性。方法采用RT2PCR技术,从肿瘤细胞株总RNA中克隆了APRIL全长编码基因;构建了APRIL胞外可溶性片段(sAPRIL)的原核表达载体,对表达的蛋白初步纯化后进行生物学活性检测;构建了APRIL编码区的真核表达载体,经脂质体转染转化细胞株,流式细胞仪和MTT法分析表达产物对细胞周期及细胞生长的影响。结果sAPRIL原核表达载体经IPTG诱导后,发现在相对分子质量(Mr)20×103处有一明显的表达条带,纯化蛋白进行初步活性测定显示其可以剂量依赖方式促进细胞的生长;细胞转染实验证实APRIL能促进多种转化细胞的增殖,但对细胞周期无明显影响。结论成功克隆了人APRIL基因,并将其可溶性胞外区片段在大肠杆菌进行了表达;构建了APRIL真核表达载体,初步分析了APRIL的生物学活性,提示APRIL在肿瘤细胞增殖过程中可能起了重要作用,为进一步进行APRIL基因的功能研究及其临床应用奠定了实验基础。
Objective To clone human proliferation inducing ligand (APRIL) gene and analyze the biological activity of APRIL protein. Methods The full-length APRIL gene was cloned from the total RNA of tumor cell lines by RT2PCR. The prokaryotic expression vector of APRIL extracellular soluble fragment (sAPRIL) was constructed. The expressed protein was purified and its biological activity was detected. APRIL coding region of eukaryotic expression vector, liposome-transfected cell lines, flow cytometry and MTT analysis of expression products on cell cycle and cell growth. Results After induced by IPTG, the prokaryotic expression vector of sAPRIL showed a clear expression band at the molecular weight of 20 × 103, and the purified protein showed that it could promote the cell growth in a dose-dependent manner. The transfection experiment Confirmed that APRIL can promote the proliferation of a variety of transformed cells, but no significant effect on cell cycle. Conclusion The human APRIL gene was successfully cloned and its soluble extracellular region was expressed in E. coli. The APRIL eukaryotic expression vector was constructed and the biological activity of APRIL was analyzed preliminarily, which indicated that APRIL may play an important role in the proliferation of tumor cells Which has laid an experimental foundation for the further study on the function of APRIL gene and its clinical application.