可调控性人PTEN基因重组腺病毒的构建与表达  

Construction and expression of regulated human tumor suppressor gene PTEN recombinant adenovirus vector

作　　者：邱镇[1] 孙颖浩[1] 许传亮[1] 沈茜[2] 钱松溪[1] 李云[1] 

机构地区：[1]第二军医大学附属长海医院泌尿外科,上海200433 [2]第二军医大学附属长海医院中心实验室,上海200433

出　　处：《中华实验外科杂志》2003年第5期453-455,共3页Chinese Journal of Experimental Surgery

基　　金：全军医学科学技术研究"十五"计划重点课题 (0 1Z0 67)

摘　　要：目的　体外构建和表达可调控性人抑癌基因PTEN重组腺病毒。方法　将人PTEN基因全长cDNA约 1.4kb经过穿梭载体 ,与可调控性腺病毒载体骨架连接 ,得到重组人PTEN基因腺病毒 (Ad PTEN) ,酶切与聚合酶链反应 (PCR)鉴定后 ,在HEK2 93细胞包装、扩增 ,空斑试验测定病毒滴度 ,逆转录 聚合酶链反应 (RT PCR)、Westernblot法分别在mRNA、蛋白水平检测PTEN的表达。Ad X TRE βgal病毒作为对照。 结果　构建的Ad PTEN病毒滴度为 1.8× 10 7pfu/ml ,转染前列腺癌细胞株PC 3后RT PCR扩增出特异条带 ( 4 62bp) ,Westernblot检测PTEN蛋白 ( 60×10 3 )有高表达 ,对照病毒转染后未测到PTEN的表达。结论　用体外连接法成功地构建了可调控性人抑癌基因PTEN重组腺病毒载体 ,并得到了正确、特异的表达。Objective To construct and express a recombinant adenovirus vector which can regulate expression of human tumor suppressor gene PTEN.Methods The full length cDNA 1.4kb encoding PTEN gene was digested from pcDNA3 PTEN plasmids and put into pTRE Shuttle vector.Then,the expression cassette containing tetracycline responsive element which can regulate the expression of inserted genes was excised by digesting with the restriction enzymes PI SceI and I CeuI and ligated to the backbone of the Adeno X Viral DNA.The recombinant adenovirus plasmid was identified by restriction enzymes excising analysis and polymerase chain reaction (PCR).The replication incompetent recombinant adenovirus which has the capability of transfection was packaged and propagated in HEK293 cell.The viral titer was examined by plaque assay and the mRNA and protein expressions of PTEN in human prostate cancer cell line PC 3 infected with Ad PTEN were determined by reverse transcription polymerase chain reaction (RT PCR) and Western blot respectively.Ad X TRE βgal virus was used as control.Results The viral titer of Adeno PTEN was 1.8×10 7 pfu/ml and the PTEN mRNA (462 bp) and protein (60 KD) expression was positive and high,whereas the titer of control virus was 1.6×10 7 pfu/ml and the PTEN protein expression was negative.Conclusion The human tumor suppressor gene PTEN recombinant adenovirus has been set up successfully and express high,which was a base of prostate cancer gene therapy.

关 键 词：前列腺癌 PTEN 基因表达 腺病毒载体 

分 类 号：R346[医药卫生—基础医学]