乳腺癌转移抑制基因BRMS1真核表达载体构建及鉴定.docVIP

乳腺癌转移抑制基因BRMS1真核表达载体构建及鉴定 　 作者：唐鲁兵 高海东 田春艳 孙靖中 刘焕涛 马榕 王甜甜 【摘要】目的：构建乳腺癌转移抑制基因（BRMS1）真核表达载体pcDNA3.1/mycBRMS1，为研究抑制恶性肿瘤转移的机制奠定基础。方法：设计含有Hind Ⅲ和XhoⅠ酶切位点的一对引物，应用逆转录聚合酶链式反应（RTPCR）技术从人乳腺癌细胞株MCF7中扩增到BRMS1的cDNA基因片断，经回收、纯化、酶切后，连接到质粒pcDNA3.1/mycHis(+)A上，鉴定正确后并转染人胚肾细胞HEK293进行Western blot验证其是否表达。结果：琼脂糖凝胶电泳及基因测序证实重组的pcDNA3.1/mycBRMS1 cDNA的碱基组成与GenBank中的人BRMS1 cDNA的基因片断组成相同。结论：成功构建了乳腺癌转移抑制基因BRMS1真核表达载体并测序鉴定。 【关键词】乳腺肿瘤#12539;转移抑制基因#12539;BRMS1#12539;基因表达 Construction and identification of eukaryotic expression vector for human breast cancer metastasis suppressor1(BRMS1) 【ABSTRACT】Objective:To construct recombined eukaryotic expression vector pcDNA3.1/mycBRMS1cDNA successfully.This is useful for researching the mechanisms of metastatic suppression.Methods:To design a pair of primers,which two restriction site with HindⅢ and XhoⅠ,and BRMS1cDNA fragments were amplified from human breastcancer MCF7 by RTPCR.The product was extracted,purified and digested with Hind Ⅲ and Xho Ⅰ,then was inserted to PcDNA3.1/mycHis(+)A plasmid.The recombinants transfected into HEK293 cell and was identified by Western blot.Results:The base sequence of recombined pcDNA3.1/mycBRMS1cNDA was in accordance with human BRMS1 cDNA fragments by agarose gel electrophoresis and DNA sequence analysis.Conclusion:Eukaryotic expression vector for human breast cancer metastasis suppressor 1(pcDNA3.1/mycBRMS1)has been constructed successfuly and DNA sequence was analyzed. 【KEY WORDS】Breast neoplasms #12539;Metastasis suppressor gene#12539;BRMS1#12539;Gene expression 与乳腺癌转移有关的基因分为转移促进基因和转移抑制基因两大类。乳腺癌转移抑制基因1（breastcancer metastasis suppressor1,BRMS1）是新发现的肿瘤转移抑制基因，同其它肿瘤转移抑制基因（Nm23,KISS1,Kail,ECadherin,MKK4,TIMPs等）一样，BRMS1并不影响原发肿瘤的生长，仅仅抑制癌细胞向远处部位转移［1］。为了进一步研究BRMS1，抑制恶性肿瘤转移的机制，我们应用RTPCR技术构建了带有BRMS1cDNA基因片段的真核表达载体PcDNA3.1/mycBRMS1。 1 材料与方法 1.1 材料 1.1.1 细胞与质粒 人乳腺癌细胞株MCF7及人胚肾细胞株HEK293为军事医学科学院放射医学研究所惠赠，质料PcDNA3.1/mycHis(+)A购自Invitrogen公司。 1.1.2 引物设计与合成 根据BRMS1cDNA序列，设计一对特异性引物，上游引物：5’agaaagcttccaccatgcctgtccagcc3’,含Hind