title: [Stable expression of recombinant human podoplanin in Chinese hamster ovary (CHO) cells].
abstract: 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 PDPN-EGFP was obtained after sorting by flow cytometry. Western blotting showed that the recombinant PDPN was expressed on the cell surface. The over-expressing PDPN-EGFP CHO cells were able to induce human platelet aggregation.
  
  
  CONCLUSIONS
  The CHO cell line with the stable and high expression of recombinant PDPN-EGFP has been constructed successfully, and it could induce platelet aggregation.
authors.affiliation: Jiangsu Institute of Hematology, Ministry of Health Key Laboratory of Thrombosis and Haemostasis, First Affiliated Hospital, Soochow University, Suzhou 215006, China.
authors.forename: Le
authors.initials: L
authors.lastname: Qu
authors.affiliation: Jiangsu Institute of Hematology, Ministry of Health Key Laboratory of Thrombosis and Haemostasis, First Affiliated Hospital, Soochow University, Suzhou 215006, China.
authors.forename: Xingpeng
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Jiangsu Institute of Hematology, Ministry of Health Key Laboratory of Thrombosis and Haemostasis, First Affiliated Hospital, Soochow University, Suzhou 215006, China.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Fu
authors.affiliation: Jiangsu Institute of Hematology, Ministry of Health Key Laboratory of Thrombosis and Haemostasis, First Affiliated Hospital, Soochow University, Suzhou 215006, China.
authors.forename: Lijun
authors.initials: L
authors.lastname: Xia
authors.affiliation: Jiangsu Institute of Hematology, Ministry of Health Key Laboratory of Thrombosis and Haemostasis, First Affiliated Hospital, Soochow University, Suzhou 215006, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Dai
authors.affiliation: Jiangsu Institute of Hematology, Ministry of Health Key Laboratory of Thrombosis and Haemostasis, First Affiliated Hospital, Soochow University, Suzhou 215006, China.
authors.forename: Changgeng
authors.initials: C
authors.lastname: Ruan
authors.affiliation: Jiangsu Institute of Hematology, Ministry of Health Key Laboratory of Thrombosis and Haemostasis, First Affiliated Hospital, Soochow University, Suzhou 215006, China. *Corresponding author, E-mail: zhaoyimingbox@163.com.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010974:Platelet Aggregation / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection; D000098853:Podoplanin
chemical_list: D049452:Green Fluorescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C117468:PDPN protein, human; D000098853:Podoplanin; C485184:enhanced green fluorescent protein