重组腺病毒rhAdEasy-nm23-H1的构建与鉴定及体外表达的实验研究

发布时间：2018-03-19 19:19

本文选题：nm23-H1　切入点：重组腺病毒　出处：《昆明医学院》2006年硕士论文　论文类型：学位论文

【摘要】：【目的】利用复制缺陷型腺病毒载体和nm23-H1基因构建出重组真核表达载体并鉴定其在体外培养的肿瘤细胞中的表达。【方法】设计带酶切位点Kpn Ⅰ，Xho Ⅰ的引物，应用PCR技术从质粒pMD18-T-nm23上克隆出nm23-H1cDNA并定向克隆至穿梭载体pShuttle-CMV，构建出重组穿梭质粒pShuttle-nm23，经过Kpn Ⅰ，Xho Ⅰ双酶切和DNA测序鉴定后，利用pShuttle-nm23和腺病毒骨架载体pAdEasy-1在细菌BJ5183菌体内进行同源重组，卡那霉素筛选，构建出重组腺病毒质粒rhAdEasy-nm23-H1，重组腺病毒质粒经Pac Ⅰ酶切鉴定和PCR鉴定后，在工程菌XL10-Gold菌体内大量扩增，CsCL梯度离心纯化重组腺病毒质粒后转染AD-293细胞，在AD-293细胞内进行腺病毒的包装扩增，测定重组腺病毒感染性滴度TCID_(50)，，行免疫荧光，免疫组化和Western blot实验以检测nm23在肺癌细胞YTMLC和GLC细胞中的表达。【结果】将nm23-H1cDNA正向连接到穿梭载体pShuttle-CMV上，Kpn Ⅰ，Xho Ⅰ双酶切能切出460bp的的片段，DNA测序结果与Genebank上nm23-H1基因编码区完全一致，该重组质粒与腺病毒骨架载体pAdEasy-1在细菌BJ5183菌体内成功进行同源重组，并经Pac Ⅰ酶切鉴定，得到带有目的基因的大小为3.0kb或4.5kb的穿梭质粒的片段，测得重组腺病毒感染性滴度为10~7TCID_(50)／mL，经免疫荧光，免疫组化和Western blot检测，nm23在肺癌细胞YTMLC和GLC里有表达。【结论】本实验成功构建出携带nm23-H1cDNA的真核表达载体重组腺病毒rhAdEasy-nm23-H1：并且用该重组腺病毒对肿瘤细胞进行转染，证明导入的nm23-H1基因可以在肿瘤细胞中表达，为下一步进行nm23抑制肿瘤细胞转移的功能鉴定打下基础。
[Abstract]:[Objective] to construct recombinant eukaryotic expression vector using replication defective adenovirus vector and nm23-H1 gene, and identify its expression in tumor cells in vitro.
[method] designed with restriction sites of Kpn I, Xho I primer, the application of PCR technology to clone nm23-H1cDNA and cloned into the shuttle vector pShuttle-CMV from plasmid pMD18-T-nm23, the recombinant shuttle plasmid pShuttle-nm23, after Kpn I, Xho I double enzyme digestion and DNA sequencing and homologous recombination in bacteria, BJ5183 bacteria the use of pShuttle-nm23 and adenovirus backbone vector pAdEasy-1, kanamycin screening, the recombinant adenovirus plasmid rhAdEasy-nm23-H1, the recombinant adenovirus plasmid by Pac I digestion and PCR assay after amplified in E.coli XL10-Gold in bacteria, CsCL purified recombinant adenovirus plasmid was transfected into AD-293 cells by adenovirus AD-293 cells within the package and amplification of recombinant adenovirus, determination of infectious titer of TCID_ (50), immunofluorescence, immunohistochemistry and Western blot experiments to detect nm23 in lung cancer Expression in cell YTMLC and GLC cells.
[results] the nm23-H1cDNA is connected to the shuttle vector pShuttle-CMV, Kpn I, Xho I double enzyme digestion can be cut out of the 460bp fragment of DNA was proved to be consistent with the Genebank encoding region of nm23-H1 gene, the recombinant plasmid and adenovirus backbone plasmid pAdEasy-1 for homologous recombination in bacteria BJ5183 successfully in bacteria, and by Pac I digestion, get a gene size is 3.0kb or 4.5kb shuttle plasmid fragment obtained recombinant adenovirus infection titer was 10~7TCID_ (50) / mL by immunofluorescence, immunohistochemistry and Western blot assay, the expression of nm23 in YTMLC and GLC lung cancer cells.
[Conclusion] this experiment successfully constructed the eukaryotic expression vector of recombinant adenovirus carrying nm23-H1cDNA and rhAdEasy-nm23-H1 of tumor cells were transfected with the recombinant adenovirus, nm23-H1 gene that can be expressed in tumor cells, and lay the foundation for the functional characterization of nm23 inhibit the metastasis of tumor cells in the next step.

【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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