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Title of Journal: Mol Biol Rep

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Abbravation: Molecular Biology Reports

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Springer Netherlands

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DOI

10.1016/0042-207x(67)90164-9

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ISSN

1573-4978

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Molecular cloning and expression of glycoprotein I

Authors: Lei Ren Limei Zhang Yanhong Liu
Publish Date: 2008/09/02
Volume: 36, Issue: 6, Pages: 1421-1425
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Abstract

Objective To construct the eukaryotic expression vectors of mutant GPIIIa establish CHO cell lines stably expressing mutant GPIIIa Methods Total RNA were extracted from HEL cells Mutant GPIIIa cDNA was synthesized by RTPCR using the specific primers designed according to Genbank by Primer 5 then leaded to T1565C The expression vector pcDNA31+ and PCR products were respectively digested by NheI and HindIII the specific cDNA fragments were directly inserted to the pcDNA31+ because of having the same adhesive ends Then wild type pcDNA31+IIIa and mutant pcDNA31+IIIa were respectively transfected into CHO cells using Lipofectamine 2000 reagent The cell lines expressing GPIIIa and GPIIIaT1565C were screened by G418 Expression of GPIIIa and GPIIIaT1565C on transfected CHO cell surface were evaluated by flow cytometry and by RTPCR to substantiate mRNA Results The cDNAs of GPIIIa and GPIIIaT1565C were amplified by RTPCR and the recombinant of mutant pcDNA31+IIIa were constructed By sequencing and enzyme digestion it was be confirmed that there is a mutant of GPIIIa on 1565T → C The result of flow cytometric analysis showed fluorescence intensity in the CHO cells transfected by recombinant is much higher than that by pcDNA31+IIIa Conclusions 1 Succeeded in constructing recombinants pcDNA31+IIIaT1565C 2 Succeeded in getting the cell lines expressing GPIIIaT1565C

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