NT_4-SAC-HA_2-TAT融合基因表达载体的构建及鉴定  

Construction and identification of the expression vector of NT_4-SAC-HA_2-TAT fusion gene

作　　者：张士宝[1] 刘庆勇[1] 阮喜云[2] 陈杰[1] 张建军[1] 李宗武[1] 杨广笑 王全颖 

机构地区：[1]山东大学附属济南市中心医院泌尿外科,济南250013 [2]山东大学附属济南市中心医院神经内科,济南250013 [3]西安华广生物工程公司,西安710025

出　　处：《山东大学学报（医学版）》2009年第6期15-19,共5页Journal of Shandong University:Health Sciences

摘　　要：目的构建NT4-SAC-HA2-TAT融合肽cDNA克隆,探讨前列腺凋亡反应基因-4(Par-4)核心区凋亡肽(SAC)作为目的基因对前列腺癌的治疗作用。方法应用互为引物模板法合成两端具有NaeI和KpnI酶识别位点的SACcDNA片段。将所得SAC和已有HA2-TAT片段克隆到具有相应酶切位点的pBV220-NT4质粒,得到pBV220-NT4-SAC-HA2-TAT重组质粒。PCR扩增NT4-SAC-HA2-TATcDNA片段,并将其克隆到pGEM-T easy中,转化细菌筛选阳性克隆,酶切鉴定,序列测定分析。结果经DNA测序、限制性内切酶酶切等证实,PCR获得了编码NaeI和KpnI酶切位点的SAC cDNA片段,并将NT4、SAC、HA2-TAT亚克隆于pBV220内。结论成功构建了NT4-SAC-HA2-TAT融合基因的原核表达载体pBV220-NT4-SAC-HA2-TAT。Objective To construct the prokaryocyte expression vector bearing fusion gene NT4-SAC-HA2-TAT and lay a foundation for further study on the genetic therapy of prostate cancer. Methods By means of asymmetrical primer/template, the fragment encoding SAC was gained by the PCR method. Then the SAC/Noel, KpnI and HA2-TAT/KpnI, and Xho I were cloned into recombinant vector pBV220-NT4 / Nae I, SalI and the recombinant plasmid pBV220-NT4 -SAC-HA2 -TAT was obtained. The fragment NT4-SAC-HA2-TAT obtained was amplified by PCR. Then the fragment was easily cloned into vector pGEM-T and was sequenced by the dideoxy-mediated chain-tennirlation method. The cloned NT4-SAC-HA2-TAT eDNA was compared with the Gene- Bank sequence by the DNASIS. Results By DNA sequencing, the sequence of SAC was obtained which included Noel and KpnI restriction enzyme sites by per. The fusion gene NT4-SAC-HA2-TAT was successhtlly sub-cloned into pBV220. Conclusion The recombinant vector pBV220-NT4-SAC-HA2-TAT was successfully constructed. These results lay the foundation for further research of using fusion genes to treat prostate cancer.

关 键 词：前列腺凋亡反应基因-4 融合肽 前列腺肿瘤 基因疗法 

分 类 号：R737.25[医药卫生—肿瘤]