人乳腺癌转移抑制基因真核表达载体的构建及鉴定

Construction and identification of eukaryotic expression vector for human breast-cancer metastasis suppressor 1 （BRMS1）

目的构建乳腺癌转移抑制基因（BRMS1）的真核表达载体pcDNA3．1（-）B／myc-BRMSI，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，Trizd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BIIMS1 cDNA的CDS序列，连接到质粒pcDNA3．1／mye—His（-）B上，构建BRMS1的真核表达载体peDNA3．1（-）B／myc．BRMS1，行基因测序鉴定正确后转染人胚肾细胞HEK-293，行Westernblot验证其是否表达。结果重组质粒pcDNA3．1（-）B／myc-BRMS1经双酶切及基因测序分析，验证了克隆的人BRMS1基因cDNA序列与GenBank[AF159141]公布的人BRMS1基因的cDNA序列吻合，重组体peDNA3．1（-）B／myc—BRMS1中插入的目的基因BRMS1是正向、单倍插入。结论成功构建了BRMS1真核表达载体pcDNA3．1（-）B／myc—BRMS1，为深入研究BRMS1基因功能和BRMS1基因治疗奠定了物质基础。

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 therapy. Methods To isolate total RNA from MCF -7 ceils and design a pair of primers,and coding sequence of BRMS 1 cDNA were amplified from human breast cancer ceils 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 BRMS1 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.