核心蛋白聚糖真核表达载体的构建及其表达  

Construction of an eukaryotic expressing vector of decorin and its expression in HCT-8 cells

作　　者：操海萍[1] 舒振波[2] 张桂珍[1] 

机构地区：[1]吉林大学中日联谊医院中心实验室 [2]吉林大学中日联谊医院胃肠外科,长春130031

出　　处：《肿瘤》2007年第5期345-347,360,共4页Tumor

基　　金：吉林省科技厅发展计划资助项目(编号:20061550)

摘　　要：目的:构建人核心蛋白聚糖(decorin,DCN)真核表达载体,并在人结直肠癌HCT-8细胞中进行表达,以研究其抗肿瘤作用。方法:以克隆有完整人DCN基因片段的质粒pBluescript为模板,采用聚合酶链式反应(PCR)扩增获得目的基因片段。真核表达载体pcDNA3及PCR产物经XbaⅠ和EcoRⅠ双酶切后连接,转化大肠杆菌JM109,获得重组载体pcDNA-DEC,进行酶切鉴定和测序鉴定。脂质体lipofectamine介导重组载体转染HCT-8细胞,经G418(800μg/mL)筛选建立稳定转染细胞株。采用免疫组化法检测转染后HCT-8细胞中DCN的表达。MTT法检测HCT-8细胞的增殖活力。结果:PCR扩增获得与预期大小一致、约1 000 bp的特异性DNA片段;重组载体pcDNA-DEC经双酶切鉴定及测序证实,人DCN基因cDNA片段正确插入真核表达载体中。免疫组化结果显示在稳定转染的HCT-8细胞株中可见DCN蛋白表达。细胞生长曲线显示转染DCN的HCT-8细胞生长较对照组减慢。结论:成功构建DCN真核表达载体pcDNA-DEC,并获得稳定转染的HCT-8细胞株,为进一步研究DCN抗肿瘤作用奠定基础。Objective ：To construct an eukaryotic expressing vector of DCN and express it in HCT-8 cells to investigate its antitumor effects. Methods： The target DCN gene fragment was amplified by polymerase chain reaction （PCR） based on the template of plasmid pBluscript containing complete human DCN gene. Eukaryotic expressing plasmid pcDNA3 and PCR product were ligated and transformed into E. coli JM109 after double digestion with Xba Ⅰ and EcoR Ⅰ to obtain the recombinant pcDNA-DEC. The DCN expression vector was verified by restrictive endonuclease digestion and DCN sequencing analysis. DCN expression vectors were transfected into HCT-8 cells by lipofectamine and stably transfected cells were selected by G418 （800 μg/mL） screening for about 2 months. The expression of DCN protein in stably transfected cells was detected by immunohistochemistry. Results： The specific DNA fragment of DCN in 1 000 bp was obtained by PCR amplification. Double restrictive enzyme digestion and DNA sequencing analysis verified that the cDNA fragment of human DCN gene was correctly inserted into the eukaryotic expressing vector. The expression of DCN protein was detectable in stably transfected HCT-8 cells. Cell growth curve analysis showed that the growth of stably transfected HCT-8 cells was slower than that of control cells. Conclusion： We successfully construct the recombinant eukaryotic expression vector of pcDNA-DEC and establish the stably transfected HCT-8 cells which provided an experimental basis for further researching of the anti-tumor effects of DCN.

关 键 词：基因扩增 基因 Decorin 转染 HCT-8细胞 

分 类 号：Q784[生物学—分子生物学]