Construction Of PcDNA3.1(+)-CLEC2 Eukaryotic Expression Vector And Expression In CHO Cells

The human CLEC-2 is a characterized C-type lectin-like receptor,which has been shown to mediate platelet activation and aggregation.CLEC-2 is signaling upon binding to rhodocytin,a snake venom protein,so it is a receptor for rhodocytin.And it is also a identified co-receptor for human immunodeficiency virus type 1(HIV-1).Therefore,we research CLEC-2,it can guide to design the drugs of anti-platelet activation and aggregation,and it provides bases for thrombotic cardiovascular disease treatment and benefits to study the spread of HIV.Objective:To construct eukaryotic expression vector pcDNA3.1(+)-CLEC2 and express CLEC-2 in CHO cells.Methods:1.The construction of the eukaryotic expression vector pcDNA3.1(+)-CLEC2:The primers of CLEC-2 gene were designed according to GenBank databases using Primer Premier 5.0 software,and 5' end of the primers was added restriction enzyme site.The total RNA was extracted from human platelet with QIAamp RNA Blood Mini Kit.CLEC-2 gene fragment was amplified using RT-PCR and then purifed with TIANgel Midi Purification Kit.After digesting CLEC-2 gene with HindⅢ和EcoRⅠ,we ligated it to the vector pcDNA3.1(+) using T4 DNA ligase.Then the recombinant clones were identified by ampicillin,and the eukaryotic expression vector pcDNA3.1(+)-CLEC2 was constructed which was identified by PCR,enzyme cut and sequencing.2.The recombinant plasmid pcDNA3.1(+)-CLEC2 transfected CHO cell:The method of lipofectamine transfection was used to transfect CHO cell with the recombinant plasmid pcDNA3.1(+)-CLEC2.The transfected cells were screened with G418 for 30 days to obtain the CHO cell line which can consistently express gene,then we detected the expression of CLEC-2 gene in protein level by Western blot. Results:1.The CLEC-2 gene was obtained through the method RT-PCR.2.We constructed successfully pcDNA3.1(+)-CLEC2 eukaryotic expression plasmid.The target fragment identified with PCR and HindⅢ,EcoRⅠdouble-enzyme cutting system was consistent with expection.The sequencing to CLEC-2 gene was consistent with GenBank databases.3.We constructed CHO cell line which can express stably CLEC-2.We detected the expression of CLEC-2 gene in protein level by Western blot.Conclusion:1.We constructed successfully CLEC-2 eukaryotic expression plasmid.2.We obtained CHO cell which can express stably CLEC-2.3.The study provided experiment basis for further purification of CLEC-2 protein and investigation of its activity.