人4-1BBL基因重组腺病毒载体的构建及其在HEK293细胞的表达  

Construction and expression of human 4-1BBL recombinant adenovirus vector in HEK293 cells

作　　者：杨明花[1] 王帅[1] 田昕[1] 王扬[1] 隋承光[1] 

机构地区：[1]中国医科大学附属第一医院肿瘤研究所,辽宁沈阳110001

出　　处：《解剖科学进展》2011年第5期445-449,共5页Progress of Anatomical Sciences

基　　金：辽宁省科技厅科学技术计划项目(2010225034)

摘　　要：目的体外构建含有人4-1BBL基因的重组腺病毒载体,并检测其在HEK293细胞中的表达。方法设计一对含有SfiI酶切位点的4-1BBL基因上下游引物,以质粒pCR4-TOPO-4-1BBL为模板,通过PCR扩增获得4-1BBL基因全序列。片段回收后经酶切处理,连接到穿梭质粒pShuttle-CMV-EGFP上,获得重组穿梭质粒pShuttle-EGFP-4-1BBL。经SfiI酶切、PCR及插入片段测序鉴定正确后,将其用I-CeuI和I-SceI双酶切处理,转移到pAdxsi载体上,得到pAdxsi-GFP-4-1BBL病毒质粒,PCR鉴定正确后,经HEK293细胞包装成复制缺陷型腺病毒Ad-4-1BBL,通过观察绿色荧光蛋白(GFP)的表达,Western blot检测目的基因人4-1BBL的表达。结果酶切鉴定和序列分析证实,重组腺病毒质粒含有人4-1BBL全长cDNA编码序列,病毒滴度为1.2×1010pfu/mL。转染HEK293细胞后荧光显微镜下观察到GFP的表达,Western blot检测可见4-1BBL蛋白的正确表达。结论人4-1BBL基因重组腺病毒载体的成功构建和在HEK293细胞中的表达,为下一步将其应用于前列腺癌的免疫治疗奠定了基础。Objective To construct the recombinant adenovirus vector contained human 4-1BBL gene and to check the target gene expression in HEK293 cells. Methods Primer containing SfiI was designed and full length 4-1BBL cDNA was obtained from the pCR4-TOPO-4-1BBL plasmid by PCR, the PCR product was digested with restrictive endonucleases SfiI, then inserted directionally into pShuttle-CMV-EGFP. The plasmid of pShuttle-EGFP-4-1BBL was lined with SfiiI, the fragment containing 4-1BBL was reclaimed and then transfected into pAdxsi vector after being digested with I-CeuI and I-SceI, the recombinant adenovirus plasmid reclaimed by PCR was transfected into HEK293 cells and packed, the expression of 4-1BBL was proved by observing the green fluorescence protein （GFP） and detected by Western blot. Results Restriction enzyme assay and DNA sequence analysis showed that human 4-1BBL cDNA had been successfully inserted into recombinant adenovirus plasmid, the titer of the recombined adenovirus 4-1BBL was about 1.2×1010 pfu/ml. Green fluorescence protein was expressed in HEK293 cells transfected by pAdxsi-4-1BBL-GFP, 4-1BBL protein was expressed efficiently in HEK293 cells by Western blot. Conclusion The successful construction and expression of recombinant adenovirus Ad-4-1BBL laid a good foundation for the immunotherapy of prostate cancer.

关 键 词：4-1BBL 腺病毒 基因表达 共刺激分子 

分 类 号：Q782[生物学—分子生物学] R737.25[医药卫生—肿瘤]