Construction and identification of eukaryotic expression vector for human breast-cancer metastasis suppressor 1(BRMS1) / 中国基层医药

ABSTRACT

Objective To construct and identify the recombinant vector pcDNA3. 1 (-) B/myc-BRMS 1 carrying breast-cancer metastasis suppressor 1 (BRMS 1) which can express in eukaryote cells and which will provide the basis for further researching the mechanisms of metastasis suppression and working on cancer metastasis gene ther-apy. Methods To isolate total RNA from MCF - 7 cells and design a pair of primers, and coding sequence of aRMS 1 cDNA were amplified from human breast cancer cells MCF -7 by reverse transcription-polymerase chain reaction (RT-PCR). Then the product was inserted to the PcDNA3. 1/myc-His (-) B plasmid. The recombined pcDNA3. 1 (-)B/myc-BRMS1 was identified by gene sequence analysis,then recombinants was transfected into HEK-293 cells and was identified by Western blot. Results The recombinant of pcDNA3.1 (-) B/myc-BRMS1 was structurally confirmed by analysis of sequencing. The inserted fragment in the vector was in the right direction and its sequence was structurally confirmed to be consistent with CDS sequence of human BRMSI cDNA that of the published data. GenBank, [AF159141]. The recombinants was transfected into HEK-293 cells ,then the cells expressed protein tagged c-myc identified by Western blot indicated it can express in eukaryote cells. Conclusion cDNA of human BRMS1 can be successfully cloned and inserted into Eukaryote-expression vector. The newly constructed vector may serve as the potential tool to conduct further comprehensive experiments in future on BRMS1 function and on gene therapy.