人CD44基因真核表达载体构建及在乳腺癌细胞中表达.docVIP

人CD44基因真核表达载体构建及在乳腺癌细胞中表达 　 作者：房新建 许文林 张徐 钱晖 陈琛 方莉莉 陈巧云 【摘要】 目的： 从人类乳腺癌耐多柔比星细胞株细胞（MCF7/Adr)中克隆CD44基因并构建CD44基因真核表达载体pcDNA3.1CD44; 将pcNDA3.1CD44稳定转染入人乳腺癌细胞株（MCF7）细胞中。方法： 从MCF7/Adr细胞中提取总RNA进行RTPCR，获得全长的cDNA模板；将含有CD44基因的cDNA模板使用限制性内切酶进行双酶切获得带有酶切位点的CD44基因,再将带有酶切位点的CD44基因经TA克隆后测序验证，然后亚克隆入真核表达载体pcDNA3.1中，双酶切鉴定后再次测序验证。脂质体法将pcDNA3.1CD44质粒转染到MCF7细胞中，48小时后RTPCR和流式细胞术检测转染前后CD44基因、蛋白在MCF7细胞的表达变化。结果： 成功从人MCF7/Adr细胞中克隆获得CD44基因并构建真核表达载体，转染后在MCF7中检测到CD44基因mRNA和蛋白水平表达上调；将上述表达CD44基因及蛋白的瞬时转染MCF7细胞经G418筛选两周后获得阳性克隆。结论： 成功克隆和建立人CD44基因真核表达质粒；pcDNA3.1CD44能在MCF7细胞中表达；在MCF7/Adr中新发现一种CD44基因的变异体(基因库号FJ216964)；这为进一步研究其基因功能打下了基础。 【关键词】 CD44； 多药耐药； 侵袭； 乳腺癌 ［Abstract］ Objective: To clone CD44 gene from multidrug resistant human mammary carcinoma cells(MCF7/Adr) and construct its eukaryotic expression vector. Which was transfected into MCF7 cells to obtain position clone.Methods： The full length cDNA encoding CD44 was obtained by RTPCR from the total RNA isolated from the MCF7/Adr cells and cloned into pMD19T vector. The CD44 gene sequence and reading frame were confirmed by two restriction enzymes and nucleotides sequencing, then inserted in the eukaryotic expression vector pcDNA3.1. The recombinant vector pcDNA3.1CD44 was transfected into MCF7 cells, and the expression changes of CD44 gene and protein were detected at 48 hour later, respectively. Results： CD44 gene was cloned from MCF7/Adr cells,and its eukaryotic expression vector was constructed successfully. After the identification and sequencing, the reconstructed plasmid was confirmed containing the sequence of CD44 gene. After transient transfection, the mRNA and protein level of CD44 gene were obviously upregulated in MCF7 cells. Screened with G418 for two weeks, positive clone was obtained from the above cells. Conclusion： The expression vector pcDNA3.1CD44 was constructed successfully and could be expressed in human mammary carcinoma cells. A new CD44 isoforms was found in MCF7/Adr cells(GeneBank