title: Molecular cloning and expression of glycoprotein IIb and IIIa.
abstract: OBJECTIVE
  To amplify the cDNA genes of GPIIb, GPIIIa, then construct the eukaryotic expression carriers of GPIIb and GPIIIa respectively, finally establish CHO cell lines stably expressing GPIIb and GPIIIa.
  
  
  METHODS
  Human erythroleukemia (HEL) cells were cultured for total RNA extraction. RT-PCR was accomplished using the specific GPIIb, GPIIIa primers designed according to Genbank by Primer 5, then each of cDNAs were obtained. The expressive vector pcDNA3.1(+) and PCR products were cut by NheI and HindIII, and then the fragements were directly cloned to pcDNA3.1(+) because of having the same adhesive ends. Then pcDNA3.1(+)IIb and pcDNA3.1(+)IIIa were transfected into CHO cells respectively by Lipofectamine 2000. The cell lines expressing GPIIb, GPIIIa were screened by G418. Then the Chinese hamster ovary (CHO) cell lines were examed through flow cytometry (FCM) and RT-PCR to detect the expression of GPIIb, GPIIIa in CHO cells.
  
  
  RESULTS
  The cDNAs of GPIIb and GPIIIa were amplidied by RT-PCR, and the pcDNA3.1(+)IIb and pcDNA3.1(+)IIIa were constructed respectively. By sequencing and double digestion, pcDNA3.1(+)IIb and pcDNA3.1(+)IIIa were all correct. Expression of GPIIb and GPIIIa were detected on transfected CHO cells by FCM and RT-PCR.
  
  
  CONCLUSIONS
  (1) Succeeded in constructing pcDNA3.1(+)IIb, pcDNA3.1(+)IIIa. (2) Succeeded in getting the cell lines expressing GPIIb, GPIIIa.
authors.affiliation: Department of Laboratory Medicine, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, P.R. China. naihanlei@126.com
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Liu
authors.forename: Lei
authors.initials: L
authors.lastname: Ren
authors.forename: Jinghong
authors.initials: J
authors.lastname: An
authors.forename: Henan
authors.initials: H
authors.lastname: Zhou
mesh_terms: D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000032:analysis; D015870:Gene Expression; D006801:Humans; D039661:Integrin beta3 / Q000235:genetics* / Q000378:metabolism; D040201:Platelet Membrane Glycoprotein IIb / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis
chemical_list: D018076:DNA, Complementary; D039661:Integrin beta3; D040201:Platelet Membrane Glycoprotein IIb; D011994:Recombinant Proteins