重组人平足蛋白基因在中国仓鼠卵巢细胞的稳定表达  

Stable expression of recombinant human podoplanin in Chinese hamster ovary(CHO) cells

作　　者：曲乐[1] 赵星鹏 傅建新[1] 夏利军[1] 戴兰[1] 阮长耿[1] 赵益明[1] 

机构地区：[1]苏州大学附属第一医院,江苏省血液研究所,卫生部血栓与止血重点实验室,江苏苏州215006

出　　处：《细胞与分子免疫学杂志》2016年第1期25-28,33,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81270593;81370617)

摘　　要：目的构建平足蛋白(PDPN)真核表达质粒PDPN-pEGFP-N1,建立稳定高表达重组人PDPN的中国仓鼠卵巢(CHO)细胞株并对其进行生物学活性研究。方法通过反转录PCR和DNA重组技术从HEK293细胞中克隆PDPN cDNA,插入带有增强型绿色荧光蛋白(EGFP)标记的真核表达载体pEGFP-N1中。通过PCR、酶切及测序等方法鉴定重组载体;进而将该重组载体转染至CHO细胞中,通过荧光显微镜检测EGFP的表达,Western blot法检测PDPN在CHO细胞中的表达,流式细胞术分选高表达PDPN的CHO细胞,运用血小板聚集实验检测分选后细胞株的生物学活性。结果 DNA测序和酶切鉴定证明PDPN基因正确克隆至pEGFP-N1载体中,重组载体稳定转染CHO细胞后,在荧光显微镜下观察到绿色荧光,流式细胞术分选后的细胞高表达PDPN;Western blot结果显示转染后CHO细胞膜表面表达PDPN蛋白;过表达PDPN的CHO细胞可以诱导人血小板聚集。结论成功构建了稳定高表达PDPN蛋白的CHO细胞株,该细胞株可表达PDPN,并诱导血小板聚集。Objective To construct podoplanin（ PDPN） eukaryotic expression plasmid PDPN-pEGFP-N1,establish Chinese hamster ovary（ CHO） cell line stably expressing recombinant human PDPN and investigate its biological activity.Methods PDPN cDNA was cloned from HEK293 cells by reverse transcription PCR and recombinant DNA technology and inserted into plasmid pEGFP-N1 labeled by enhanced green fluorescent protein（ EGFP）. The recombinant vector was identified by PCR,restriction enzyme digestion and DNA sequencing,and then transfected into CHO cells. Recombinant PDPN-EGFP was observed by fluorescent microscopy and CHO cell line with the high expression of PDPN-EGFP was selected by flow cytometry. Recombinant PDPN was detected by Western blotting and the biological activity of the cell line was determined by platelet aggregation assay. Results DNA sequencing and restriction enzyme digestion proved that the gene of PDPN was inserted successfully into pEGFP-N1 plasmid. After stable transfection of the recombinant plasmid into CHO cells,CHO with EGFP could be seen under a fluorescent microscope. The CHO cell line with the high expression of recombinant PDPNEGFP was obtained after sorting by flow cytometry. Western blotting showed that the recombinant PDPN was expressed on the cel surface. The over-expressing PDPN-EGFP CHO cells were able to induce human platelet aggregation. Conclusion The CHO cell line with the stable and high expression of recombinant PDPN-EGFP has been constructed successfully,and it could induce platelet aggregation.

关 键 词：平足蛋白 真核表达 PEGFP-N1 CHO细胞 血小板聚集 

分 类 号：R446.62[医药卫生—诊断学] Q786[医药卫生—临床医学]