摘要
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 a proliferation-inducing ligand （APRIL） gene, and to analyze its bioactivity in different cell lines, Methods The intact APRIL gene was amplified by RT-PCR using RNA isolated from tumor cell lines and its eDNA fragment was cloned into pGEM-T vector and then sequenced. The extracellular soluble fragment of APRIL（sAPRIL） was subcloned into the pET110/D-TOPO, and the bioactivity of purified sAPRIL protein was assayed by MTT methed. The APRIL open reading frame （ORF） fragment was subcloned into pcDNA3.1/V5-His-TOPT, transfected by LipofecAMINE2000 into different cell lines, and the effect of APRIL protein on cell cycle and cell growth were analyzed by cytometry and MTT assay respectively. Results sAPRIL protein with molecular mass（ Mr ） about 20 kDa was expressed in E. coli BL21 as expected after induction by IPTG, and the bioactivity analysis showed that purified sAPRIL protein significantly stimulated the growth rate of transformed ceils such as U937, Raji and Jurkat cell in a dose-dependent manner, among which Jurkat cell was the most sensitive one. The cell transfection experiment showed that transfection of APRIL expression vector could also strongly stimulate the growth of transformed ceils, but had no influence on the cell cycle as compared with those without APRIL transfection. Conclusion APRIL may play a significant role in the process of tumor ceils proliferation, which provided the possibility and basis for further research of APRIL＇s function and its clinical application.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2005年第11期907-911,共5页
Chinese Journal of Microbiology and Immunology
