摘要
目的:筛选在CHO-K1中高表达sPDGFRα-Fc的重组细胞株,并对分泌到培养基的表达产物进行抑制细胞增殖的活性分析。方法:构建带有Fc标签的sPDGFRα基因重组表达载体pIRES-Neo3-sPDGFRα-Fc;在脂质体介导下,转染CHO-K1细胞,G418筛选2周后获得若干单克隆细胞株,随机挑取单克隆细胞进一步放大培养,RT-PCR筛选阳性单克隆细胞;Real-Time PCR方法鉴定各阳性细胞株中的sPDGFRα-Fc基因的转录水平,Western blot检测进一步验证各细胞中目的蛋白表达水平;筛选出表达最高的细胞株,更换无血清培养基培养,取含有可溶性sPDGFRα的培养基上清冻干浓缩,MTT法检测目的蛋白的抑制细胞增殖能力。结果:成功构建重组表达载体并在CHO-K1中成功表达,各阳性单克隆细胞株的表达量有差异且在转录水平和蛋白表达水平表现一致,从无血清培养基中收集的可溶性sPDGFRα-Fc明显抑制血管内皮细胞的增殖。结论:成功筛选获得CHO-K1中高表达sPDGFRα-Fc的重组细胞株,获得的可溶性sPDGFRα-Fc能抑制细胞增殖,有望成为治疗因PDGF及其受体引起的多种疾病的药物。
Orjective： To obtain recombinant CHO-K1 with expressing sPDGFRα and to identify the biological activities of sPDGFRα secreted in non-serum medium. Methods： Recombinant human sPDGFRα expression vector pIRES-Neo3-sPDGFRα-Fc was constructed and then transfected into CHO-K1 cells by using LipofectamineTM 2000. After screened with G418 in 8 weeks, some monoclone cells were selected randomly to amplify in 96-well-plate to 24-well-plates, and then to identify positive cell clones by RT-PCR. Furthermore, the candidate cell clones were test by Real-Time PCR and Western blot assays. Finally, anti-proliferation activities of the expressed sPDGFRα were analyzed by M3T. Results： sPDGFRα-Fc was cloned into pIRES-Neo3 correctly. The sPDGFRα-Fc expression level in recombinant CHO-K1 cell clones were concordant in between Reahime PCR and Western blot assay, sPDGFRα-Fc obtained from cultured non-serum medium of positive CHO-K1 could significantly inhibit proliferation of vascular endothelial cell. Conclusion：Successed to select recombinant CHO-K1 cell lines with high expressed sPDGFRα-Fc. The sPDGFRα-Fc can inhibit the cell proliferation significantly and it means sPDGFRα-Fc might be a new anti-cancer drug in the future.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2009年第7期1-6,共6页
China Biotechnology
基金
国家"863"计划重大专项子课题(2002AA22344E)
广东省科技攻关项目(200813030301349)资助项目
