美洲商陆抗病毒蛋白在毕赤酵母中的表达及其诱发人神经胶质瘤细胞U251凋亡的研究  

Cloning and Expression of Pokeweed Antiviral Protein Gene from Phytolacca amercana in Pichia pastoris and the Study of Apoptosis of Human Neuroglioma Cells U251 Induced by Recombinant PAP

作　　者：向莉[1] 胡亚梅[1] 张杰文[1] 

机构地区：[1]河南省人民医院神经内科,郑州450003

出　　处：《中国中西医结合杂志》2011年第8期1104-1107,共4页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：河南省科技攻关项目(No.092102310084)

摘　　要：目的研究美洲商陆抗病毒蛋白(pokeweed antiviral protein,PAP)基因的克隆表达,进而研究其诱发人神经胶质瘤细胞U251凋亡。方法利用RT-PCR技术克隆PAP基因,构建PAP毕赤酵母表达质粒pPICZaA-PAP并导入毕赤酵母Pichia pastorisGS115。SDS-PAGE检测PAP的分泌表达。采用镍离子亲合层析纯化PAP,并通过单细胞凝胶电泳和MTT检测其抑制人神经胶质瘤细胞U251的生长。结果分泌表达的PAP融合蛋白分子量约为35/kD,纯化的PAP在体外能诱发人神经胶质瘤细胞U251凋亡,PAP对U251半数抑制浓度(IC50)为81.0μg/mL,通过单细胞凝胶电泳,能看到明显的慧星尾,表明PAP引起了神经胶质瘤细胞基因组的降解。结论 PAP蛋白能抑制神经胶质瘤细胞的生长及诱发人神经胶质瘤细胞U251凋亡。Objective To clone the pokeweed anti-viral protein （PAP） gene,to express it in Pichia pastroris,and to study the inhibitory effect of PAP on U251 in vitro.Methods The cDNA sequence encoding PAP was cloned by Real-time PCR from Phytolacca amercana.The recombinant PAP was subcloned into the expression vector pPICZaA and expressed in Pichia pastroris GSI15 after methanol induction.SDS-PAGE analysis showed that the expressed PAP existed in the yeast culture supernatant.The drug cytotoxicity to U251 cells was assessed using MTT assay and the obvious apoptotic nuclei of the tumor cells detected using the method of single cell gel electrophoresis.Results The full-length PAP gene was cloned.The recombinant expression plasmid pPICZaA-PAP was constructed successfully.SDS-PAGE analysis showed that the relative molecular mass （M） of the recombinant protein was about 35 kDa.The degradation of the genome of the apoptotic cells induced by PAP was detected using the method of single cell gel electrophoresis.PAP possessed very high ability to inhibit the growth of U251.The anti-tumor activities （IC50） to U251 cells of PAP was 81.0 μg/mL.Conclusion PAP could be a potent anti-tumor candidate for inhibiting the growth of U251 and inducing its apoptosis.

关 键 词：美洲商陆抗病毒蛋白 毕赤酵母 单细胞凝胶电泳 

分 类 号：R739.4[医药卫生—肿瘤]