Construction Of Human Osteoclast Differentiation Factor Vector And Study Of It Preliminary Expression

Osteoclast differentiation factor ( ODF) is the newly discovered factor which play an important role in regulating the metabolism of bone. It have become the focus of bone metabolism. ODF has complicated effection. it is not clear that what the mechanism of ODF in bone metaboLism and ODF produced in genetic engineering provide the materiaL foundation for researching the effect and regulation of ODFunder the physiologic and condition and for establishing the means of disease test diagnose and theraty.In the experiment, we cloned and expressed the insoluble functional fragment of ODF(aal45-317) in E. coli and mammalian cells. The protein is very soon obtained in E. coli .On the other hand, the mammalian cells can express excellent functional protein which meet with the researchrequiring.In the prokaryotic cells, the sODF was amplified by PCR and inserted into the muticlone site of the expression vector pET42a. After assayed by enzyme digestion and PCR sequence, the recombined fusion expression vector pET42a/ODF was transformed into E. coli BL21 (DE3). The supersonic lycate which are assayed on SDS-PAGE geL include the recombined fusion ODF protein. Wherther The protein has the immunogenicity of human ODF verified by Western Blot analysis. It can apply to produce the antibody for the ODF.In mammalian cells, the secretive signal sequence and translation enhancing sequence were linked on the 5' of ODF cDNA by the means of PCR in order to obtaining the high expression level and avoiding the poison of the ODF to the cell. The fragment is ligated with the eukaryotic expression vector pcDNAS. 1/CT-GFP-TOPO to form the recombinant expression vector pcDNA/asODF-GFP.Furthermore, the three fragments including promoter of SV40 early gene, mouse dihydrofoLate reductase(dhfr) cDNA and SV40 polyadenylation sequence were amplified with PCR, respectively. Then, the three fragments were linked to form one fragment through the paired bases between up-stream primers and down-stream ones. After that, the wholefragment was ligated with the pcDNAS. 1/CT-GFP to form the recombinant expression vector pcDNA/DHFR-GFP.The two vectors were then cotransfected into CHO-dhfr" cells with LipofectAMINE 2000. After the selection by HT" culturing, stepwise MTX pressure culture was carried out in order to gain the high level expression clony. The integration and expression of recombinant vectoris assayed by RT-PCR and PCR whose template is from the RNA and genome DNA which are extract from the transfected cells.