Construction And Expression Of Eukaryotic Expression Vectors Of Human Wild-Type Cited2and Mutant-Type Cited2

Objective To construct recombinant eukaryotic vectors expressing wild-type CITED2and mutant-type CITED2of human transcriptional coactivator, and detect the expression of CITED2protein.Methods Coding strands of CITED2, including wild-type CITED2and the mutant-type CITED2(heterozygous mutation), were obtained by PCR from blood simples of children with healthy children and congenital heart disease which we collected in our early study. The mutant point of CITED2in our study is one of we have found,which is c.573-578de16, resulted in the change of its protein (p. Ser192fs). The Coding strands of mutant-type CITED2and wild-type CITED2were connected with pMD19-T simple vector by T/A clone, which combined new vectors pMD19-T-mutant-type CITED2and pMD19-T-wild-type CITED2, by sequencing these two new vectors,we found recombinant vectors including the coding strands of mutant-type CITED2(c.573-578de16) and wild-type CITED2. Digestion of these recombinant vectors, mutant-type CITED2(c.573-578de16) and wild-type CITED2were inserted into eukaryotic expression vector pEGFP-C1. wild-type CITED2were inserted into eukaryotic expression vector pEGFP-C1, and transfected into HEK293cells. The transfected condition of pEGFP-C1-wtCITED2and pEGFP-C1-mtCITED2were observed by fluorescence microscopy. The transfected efficiency of these recombinant eukaryotic expression vectors were determined by flow cytometry. The expression of CITED2was analyzed by western blotting.Results Recombinant eukaryotic expression vectors were constructed and transfected into HEK293,24hours later, EGFP was respectively observed by fluorescence microscopy in HEK293with transfected,48hours later, the fusion expression of CITED2and EGFP were detected by western blotting. Conclusions The recombinant eukaryotic vectors expressing wild-type CITED2and mutant-type CITED2of human transcriptional coactivator were constructed successfully.