Molecular cloning and expression of glycoprotein IIb and IIIa.
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.
Liu
Yanhong
Y
Department of Laboratory Medicine, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, P.R. China. naihanlei@126.com
Ren
Lei
L
An
Jinghong
J
Zhou
Henan
H
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
D018076:DNA, Complementary; D039661:Integrin beta3; D040201:Platelet Membrane Glycoprotein IIb; D011994:Recombinant Proteins