结直肠癌细胞株增殖诱导配体基因的克隆、表达及生物学活性分析

目的克隆结直肠癌细胞株增殖诱导配体(APRIL)基因,并测定APRIL胞外可溶性片段(sAPRIL)重组蛋白的生物学活性。方法采用RT-PCR技术,从肿瘤细胞株总RNA扩增APRIL全长及其胞外可溶性片段(sAPRIL)编码基因,PCR产物直接克隆于pGEM-T载体。将sAPRIL亚克隆到原核表达载体pET101/D-TOPOR○中,阳性克隆经IPTG诱导,SDS-PAGE分析sAPRIL的表达,对表达的蛋白初步纯化后进行生物学活性检测。结果 RT-PCR扩增出一个753bpDNA片段,酶切和测序证实该片段为编码人sAPRIL的全长cDNA,将sAPRIL克隆到表达载体经诱导后发现在20kDa处多显示出一条条带,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。结论成功克隆了APRIL基因,并将其可溶性胞外区片段在大肠杆菌进行了表达。纯化的重组蛋白可促进细胞生长,为进一步进行April基因的功能研究及其开发和临床应用奠定了基础。

Cloning, expression and bioactivity analysis of a proliferation-inducing ligand of cancer cells

Objective To test the bioactivity of recombinant protein of the extracellular soluble fragment of APRIL(sAPRIL),APRIL gene cloned and expressed in Escherichia coli.Methods The intact APRIL gene was amplified by RT-PCR using RNA isolated from tumor cell lines.cDNA fragment was cloned into pGEM-T vector and sequenced.Then the sAPRIL fragment was amplified by PCR from the full length APRIL plasmid template,which was subcloned into the pET110/D-TOPO,expressed in E.coli BL21,purified and confirmed by Western blot.The bioactivity of purified sAPRIL protein was assayed by MTT method.Results A 753bp cDNA was amplified by RT-PCR and the sequence was identical with APRIL cDNA reported.The sAPRIL fragment was subcloned into the expression vector pET110/D-TOPO.After induction,a protein with molecular weight of about 20kDa was expressed as expected.Purified sAPRIL protein significantly stimulated the growth rate in transformed cells such as SW480,HT29 and Lovo cell,among which Lovo cell was the most sensitive one.Conclusions APRIL cDNA is successfully cloned and expressed,its bioactivity analysis show the purified sAPRIL protein can strongly stimulate the growth of transformed cells,which provides the possibility for further research of APRIL and basis of development and clinic research.