摘要
目的:构建前列腺特异性表达的人前列腺特异膜抗原(PSMA)基因启动子/增强子表达载体,并进行组织特异性鉴定。方法:从人外周血中提取基因组DNA,采用PCR技术分别扩增PSMA上游1175bp的启动子序列,及第三内含子中258bp的增强子序列,将两个序列定向克隆至荧光素酶报告基因表达质粒pGL3-Basic,构建前列腺特异性表达载体pGL3-PSMP-PSME。将构建载体用脂质体分别转染前列腺癌PC-3M细胞株及4种非前列腺癌细胞株,48h后通过检测荧光素酶表达活性,确定克隆的启动子及增强子的活性及其组织特异性表达活性。结果:构建的pGL3-PSMP-PSME质粒经酶切及DNA测序分析鉴定,证实克隆片段的大小、插入方向及其序列正确。细胞转染结果显示,pGL3-PSMP-PSME在PC-3M细胞株中表达活性明显高于其它4种非前列腺癌细胞株。结论:构建的前列腺表达载体具有较高的组织特异性,为进一步研究PSMA调控序列驱动治疗基因进行前列腺癌的靶向生物治疗奠定了基础。
To construct a prostate - specific expression vector of promoter and enhancer of human prostate -specific membrane antigen （PSMA）. METHODS： The promoter and enhancer of PSMA were amplified by PCR separately. The two segments were cloned into the expression vector pGL3 - Basic, a prostate - specific expression vector pGL3 -PSMP -PSME was constructed. The vector was transfected into prostatic carcinoma cell PC -3M and four kinds of non - prostatic carcinoma cells by lipofectamine. The activity of luciferase of transfected cells and tissue specificity of vector was examined at 48 hours after transfection. RESULTS： With DNA sequence of the vector pGL3 - PSMP - PSME, the segment of clone was proved correct. The activity of luciferase of the pGL3 - PSMP - PSME was expressed distinctly in PC - 3 M and was not expressed in other nonprostate cell lines. CONCLUSION： The prostate- specific expression vector was constructed successfully. It lays foundation for studying target gene therapy of the prostate cancer.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2007年第1期120-123,共4页
Chinese Journal of Pathophysiology
基金
山东省卫生厅基金资助项目(No.2003HZ042)
关键词
前列腺肿瘤
前列腺特异抗原
Prostatic neoplasms
Prostate - specific antigen
