Construction And Expression Of Sika Deer Anxa-1 Gene's Eukaryotic Expression Vector

Annexin I?Anxa-1?is a Ca²⁺-dependent phospholipid-binding protein and is a member of the annexin protein superfamily,which was first discovered and studied.Anxa-1 contains four conserved domains,and the evolution among species is relatively conservative.It is closely related to many important cellular life activities such as inflammatory response,differentiation,proliferation,death and apoptosis of cells.The expression disorder of Anxa-1 is also associated with the occurrence and development of tumors,and its expression patterns were different in the tumor cells of different oigans.In the deer antler tissue,the cell proliferation rate is faster than that of cancer cells.Although normal growth of deer antler occurs at an astonishing rate,it doesn't turn into cancer cells,so the antler tissue is also an ideal model for the rapid proliferation and development of mammalian organization.In this present study,according to the research of Qu Haomiao we designed the specific primers of Anxa-1 gene.The antler tip tissue cDNA of sika deer was used as a template,PCR was used to amplificate the Anxa-1 gene sequence of sika deer.The PCR prodct was then ligated into PMD18-T vector and transformed into competent cells of E.coli strain DH5?.The bacteria were identified by PCR,sequencing and double enzyme digestion.The sequencing results were analyzed on NCBI.The size of open reading frame of Anxa-1 gene was 1041 bp,and the length of coding amino acid was 346,which was consistent with the gene sequence obtained by Qu Haomiao.The Anxa-1 gene containing the double enzyme digestion sites was ligated into the eukaryotic expression vector pEGFP-C1,and transformed into DH5? cells.The detecting results showed that the pEGFP-C1-Anxa-1 eukaryotic expression vector was successfully constructed.pEGFP-C1-Anxa-1 was transiently transfected into 293 T cells.After 24 hours,the cells were observed under fluorescence microscope.Green fluorescence was observed.The expression level of Anxa-1 in the experimental group was more than 200 times higher than that in the control group by real-time PCR.This result indicated that pEGFP-C1-Anxa-1 eukaryotic expression vector was successfully expressed in 293 T cells.Real-time PCR was used to detect the relative expression levels of ?-catenin gene,TGF-?1 gene and Pdgfa gene,which related to Anxa-1 in regulation of cell growth and development.The expression of ?-catenin gene was significantly down-regulated?P <0.05?,and the expression of TGF-?1 gene was significantly increased?P <0.01?,but the expression of Pdgfa gene did not change significantly.