CAJ | 학술논문

目的构建乳腺癌转移抑制基因(BRMS 1)的真核表达载体pcDNA3.1(-)B/myc-BRMS1,为进一步研究恶性肿瘤的转移机制及基因治疗提供物质基础.方法常规方法培养人乳腺癌细胞株MCF-7,Trizol法提取细胞株总RNA;设计一对特异性引物,经过逆转录反应获得BRMS1 cDNA的CDS序列,连接到质粒pcDNA3.1/myc-His(-)B上,构建BRMS 1的真核表达载体pcDNA3.1(-)B/myc-BRMS1,行基因测序鉴定正确后转染人胚肾细胞HEK-293,行Western blot验证其是否表达.结果重组质粒pcDNA3.1(-)B/myc-BRMS 1经双酶切及基因测序分析,验证了克隆的人BRMS 1基因cDNA序列与GenBank[AF159141]公布的人BRMS 1基因的cDNA序列吻合,重组体pcDNA3.1(-)B/myc-BRMS 1中插入的目的基因BRMS 1是正向、单倍插入.结论成功构建了BRMS 1真核表达载体pcDNA3.1(-)B/myc-BRMS 1,为深入研究BRMS 1基因功能和BRMS 1基因治疗奠定了物质基础.
목적 구건유선암전이억제기인(BRMS 1)적진핵표체재체pcDNA3.1(-)B/myc-BRMS1,위진일보연구악성종류적전이궤제급기인치료제공물질기출.방법 상규방법배양인유선암세포주MCF-7,Trizol법제취세포주총RNA;설계일대특이성인물,경과역전록반응획득BRMS1 cDNA적CDS서렬,련접도질립pcDNA3.1/myc-His(-)B상,구건BRMS 1적진핵표체재체pcDNA3.1(-)B/myc-BRMS1,행기인측서감정정학후전염인배신세포HEK-293,행Western blot험증기시부표체.결과 중조질립pcDNA3.1(-)B/myc-BRMS 1경쌍매절급기인측서분석,험증료극륭적인BRMS 1기인cDNA서렬여GenBank[AF159141]공포적인BRMS 1기인적cDNA서렬문합,중조체pcDNA3.1(-)B/myc-BRMS 1중삽입적목적 기인BRMS 1시정향、단배삽입.결론 성공구건료BRMS 1진핵표체재체pcDNA3.1(-)B/myc-BRMS 1,위심입연구BRMS 1기인공능화BRMS 1기인치료전정료물질기출.
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.