兰州大学机构库 >学院待认领
共表达自杀基因联合多细胞因子基因的膀胱肿瘤特异性真核表达载体的构建和鉴定
Alternative TitleConstruction and Identification of Tissue Specific Eukaryotic Expression Vector for Suicide Gene Combined with Cytokine Gene
邹源
Thesis Advisor田俊强
2013-05-22
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword膀胱肿瘤 UPII启动子 单纯疱疹病毒胸腺激酶基因 白细胞介素2 肿瘤坏死因子ɑ 肿瘤坏死因子相关凋亡诱导配体 真核表达载体
Abstract目的:构建五种具有膀胱肿瘤特异性的双启动子重组真核表达载体,每种载体皆包含尿路斑块蛋白2(Uroplakin II,UPII)启动子控制的单纯疱疹病毒胸腺激酶基因(herpes simplex virus thymidine kinase,HSV-TK)、并分别搭配人白细胞介素2(interleukin-2,IL-2)、肿瘤坏死因子ɑ(tumor necrosis factor alpha,TNF-ɑ)、肿瘤坏死因子相关凋亡诱导配体(Tumor Necrosis Factor-related Apoptosis-inducing,TRAIL)、融合基因IL-2-TNF-ɑ、融合基因IL-2-TRAIL。 方法:以实验室保存的各种质粒为模板通过常规聚合酶链式反应技术(Polymerase Chain Reaction,PCR)分别扩增出UPII启动子及HSV-TK基因、IL-2基因、TNF-ɑ基因、TRAIL基因。用重叠延伸拼接技术将IL-2分别与TNF-ɑ、TRAIL融合为融合基因IL-2-TNF-ɑ(IT)、IL-2-TRAIL(ITR)。以上PCR所得各基因片段分别连接到真核载体pMD18-T并转化至感受态细胞TOP10F’中,摇菌并提取质粒。质粒经PCR和酶切鉴定为阳性后进行测序,测序结果与基因序列同源比较,选取正确的质粒进行载体的构建。用分子生物学技术将双启动子真核表达载体pBudCE4.1的EF-1ɑ启动子替换为UPII启动子形成重组载体pBud-UPII/CMV,转化至TOP10F’后摇菌并提取质粒进行鉴定,鉴定表明替换成功后在UPII后克隆入TK基因形成新载体pBud-UPII-TK/CMV并鉴定。选取鉴定结果为阳性的质粒并在pBud-UPII-TK/CMV的另一启动子CMV启动子后分别克隆入IL-2基因、TNF-ɑ基因、TRAIL基因、融合基因IT、融合基因ITR,从而构建出五种双启动子重组真核表达载体pBud-UPII-TK/CMV-IL-2、pBud-UPII-TK/CMV-TNF-ɑ、pBud-UPII-TK/CMV-TRAIL、pBud-UPII-TK/CMV-IT、pBud-UPII-TK/CMV-ITR。五种载体均转化至至感受态细胞TOP10F’中,摇菌并提取质粒,质粒进行PCR和酶切鉴定。 结果:成功克隆出UPII启动子以及人白细胞介素2(IL-2)、肿瘤坏死因子ɑ(TNF-ɑ)、肿瘤坏死因子相关凋亡诱导配体(TRAIL)、融合基因IL-2-TNF-ɑ、融合基因IL-2-TRAIL。PCR和酶切的电泳结果显示pBudCE4.1载体中的EF-1ɑ成功地被替换为UPII启动子,说明载体pBud-UPII/CMV构建成功。五种双启动子重组真核表达载体pBud-UPII-TK/CMV-IL-2、pBud-UPII-TK/CMV-TNF-ɑ、pBud-UPII-TK/CMV-TRAIL、pBud-UPII-TK/CMV-IT、pBud-UPII-TK/CMV-ITR鉴定结果均为阳性。 结论:共表达自杀基因联合多细胞因子基因的五种泌尿组织特异性真核表达载体构建成功,为今后在膀胱肿瘤细胞中表达各目的基因,并验证不同基因的抗膀胱肿瘤作用奠定了实验基础。 关键词:膀胱肿瘤, UPII启动子,单纯疱疹病毒胸腺激酶基因,白细胞介素2,肿瘤坏死因子ɑ,肿瘤坏死因子相关凋亡诱导配体,真核表达载体
Other AbstractObjective To construct five kinds of double promoter eukaryotic expression vectors,each contains HSV-TK gene droved by UPII promoter and combine with IL-2、TNF-ɑ、TRAIL、IL-2-TNF-ɑ、IL-2-TRAIL respectively. Methods UPII cDNA、TK cDNA、IL-2 cDNA、TNF-ɑ cDNA、TRAIL cDNA were amplified by polymerase chain reaction(PCR). Two fusion genes IL-2-TNF-ɑ(IT)、IL-2-TRAIL(ITR) were respectively constructed by overlap-PCR. All of the gene fragments were subcloned into pMD18-T vector.The recombined plasmids were transformed into competent cell TOP10F’. Identify the recombinants by PCR、digestion and DNA sequencing.Correct sequencing will be subcloned in pBudCE4.1 in which two strong promoters CMV and EF-1ɑ were employed. EF-1ɑ promoter was replaced with UPII promoter,and then a new vector named pBud-UPII/CMV was constructed. The restriction enzymes were employed for digestion to verify the validity of pBud-UPII/CMV. TK was subcloned in the backward position of UPII promoter and the new recombined plasmid was named pBud-UPII-TK/CMV. IL-2、TNF-ɑ、TRAIL、IT、ITR were subcloned in the backward position of CMV promoter respectively. The five new eukaryotic expression vectors pBud-UPII-TK/CMV-IL-2、pBud-UPII-TK/CMV-TNF-ɑ、pBud-UPII-TK/CMV-TRAIL、pBud-UPII-TK/CMV-IT、pBud-UPII-TK/CMV-ITR were constructed.These recombined vectors were transformed into competent cell TOP10F’ respectively,and then were identificated by PCR and digestion. Results PCR、digestion and DNA sequencing showed that the amplification of UPII promoter、TK、IL-2、TNF-ɑ、TRAIL was successful. The results of PCR and digestion showed that the EF-1ɑ promoter of pBudCE4.1 was replaced with UPII promoter,so the construction of pBud-UPII/CMV was successful. The identification of pBud-UPII/CMV、pBud-UPII-TK/CMV-IL-2、pBud-UPII-TK/CMV-TNF-ɑ、pBud-UPII-TK/CMV-TRAIL、pBud-UPII-TK/CMV-IT was positive result. Conclusion Five kinds of double promoter eukaryotic co-expression vectors (pBud-UPII/CMV、pBud-UPII-TK/CMV-IL-2、pBud-UPII-TK/CMV-TNF-ɑ、pBud-UPII-TK/CMV-TRAIL、pBud-UPII-TK/CMV-IT) were successfully constructed. This research achievement has prepared the experimental ground for the further study,which could detect the expression of vectors in bladder cancer cells and these vectors’effect on the treatment of bladder cancer. Key words bladder tumor,UPII promoter,HSV-TK,IL-2,TNF-ɑ,TRAIL ,Eukaryotic expression vector
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/223405
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
邹源. 共表达自杀基因联合多细胞因子基因的膀胱肿瘤特异性真核表达载体的构建和鉴定[D]. 兰州. 兰州大学,2013.
Files in This Item:
There are no files associated with this item.
Related Services
Recommend this item
Bookmark
Usage statistics
Export to Endnote
Altmetrics Score
Google Scholar
Similar articles in Google Scholar
[邹源]'s Articles
Baidu academic
Similar articles in Baidu academic
[邹源]'s Articles
Bing Scholar
Similar articles in Bing Scholar
[邹源]'s Articles
Terms of Use
No data!
Social Bookmark/Share
No comment.
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.