携带IKK2dn基因重组腺病毒载体的构建及表达验证

发布时间：2018-05-06 01:16

本文选题：IKK2dn + 基因转移　；参考：《苏州大学》2008年硕士论文

【摘要】： 目的: (1)构建用于真核细胞表达的重组腺病毒载体pAdxsi-GFP-IKK2dn;(2)将其包装成腺病毒颗粒,并进一步转染哺乳细胞hela并验证其在细胞中表达,为进一步研究转IKK2dn基因在同种异体器官移植中诱导免疫耐受,保护移植物的作用奠定基础。方法: (1)将pACCMVpLpASR(+)-IKK2dn基因与pShuttle-GFP-CMV(-)TEMP重组穿梭载体经KpnI/HindIII酶切,回收和纯化。T4DNA连接酶进行连接后转化为感受态细胞DH5a,丁胺卡那霉素筛选。提取单克隆菌落,作酶切鉴定,抽取阳性菌落即得到pShuttle-GFP-CMV(-)TEMP-IKK2dn重组穿梭载体质粒。(2)将重组穿梭载体质粒pShuttle-GFP-CMV(-)TEMP-IKK2dn与重组腺病毒载体质粒pAdxsi经I-CeuI+I-SceI双酶切处理,回收和纯化。T4DNA连接酶进行连接后转化为感受态细胞DH5a,氨苄青霉素筛选。提取单克隆菌落,作酶切鉴定,抽取阳性菌落即得到pAdxsi-GFP-IKK2dn病毒质粒以用于病毒包装。(3)脂质体法将重组腺病毒载体pAdxsi-GFP-IKK2dn和Lipofectamine2000脂质体共转染于人胚肾细胞系的293T细胞。观察细胞出毒迹象并进行包装,收毒,冻融,扩增,再收毒并作毒种鉴定后以离心,透析方法纯化腺病毒进行滴度测定及检测。(4)并将腺病毒颗粒转染哺乳细胞hela并验证其在细胞中表达。结果: (1)得到7.4kbp的Shuttle-GFP-CMV(-)TEMP-IKK2dn重组穿梭载体质粒。(2)腺病毒载体pAdxsi-GFP-IKK2dn序列测定结果正确。(3)最后获得的腺病毒载体滴度为2x1011 PFU/ml。(4) RT-PCR验证腺病毒颗粒成功转染哺乳细胞hela,可供进一步实验研究需要。结论: (1)成功构建Shuttle-GFP-CMV(-)TEMP-IKK2dn重组穿梭载体质粒。(2)成功构建携带IKK2dn基因重组腺病毒载体pAdxsi-GFP-IKK2dn。(3)重组腺病毒载体pAdxsi-GFP-IKK2dn和Lipofectamine2000脂质体成功包装成高滴度腺病毒颗粒。(4)腺病毒颗粒成功转染哺乳细胞hela,为进一步研究转IKK2dn基因在同种异体器官移植中诱导免疫耐受,保护移植物提供了理论和实验基础。
[Abstract]:Objective: to construct a recombinant adenovirus vector pAdxsi-GFP-IKK2dn2 for eukaryotic cell expression. The recombinant adenovirus vector pAdxsi-GFP-IKK2dn-2 was packaged into adenovirus granules and transfected into lactation cells hela and verified its expression in the mammalian cells. The results provide a basis for further study on the role of IKK2dn gene in inducing immune tolerance and protecting grafts in allogeneic organ transplantation. Methods: the recombinant shuttle vector pACCMVpLpASRN and pShuttle-GFP-CMV(-)TEMP was digested by KpnI/HindIII, and then purified by KpnI/HindIII. The recombinant plasmid was transformed into a receptive cell line DH 5a, and was screened with amikacin. The recombinant shuttle vector of pACCMVpLpASRN was digested by KpnI/HindIII. The recombinant shuttle vector plasmid pShuttle-GFP-CMV(-)TEMP-IKK2dn and the recombinant adenovirus vector pAdxsi were digested by I-CeuI I-SceI, and the recombinant shuttle vector pShuttle-GFP-CMV(-)TEMP-IKK2dn and the recombinant adenovirus vector pAdxsi were digested by I-CeuI I-SceI, respectively, and the recombinant shuttle vector plasmid pShuttle-GFP-CMV(-)TEMP-IKK2dn and the recombinant adenovirus vector plasmid pAdxsi were digested by I-CeuI I-SceI, and the recombinant shuttle vector plasmid pShuttle-GFP-CMV(-)TEMP-IKK2dn and recombinant adenovirus vector plasmid pAdxsi were digested by I-CeuI I-SceI. The DNA ligase of .T4 was recovered and purified, and then transformed into the competent cell line DH 5a for screening ampicillin. Monoclonal colonies were extracted and identified by enzyme digestion. PAdxsi-GFP-IKK2dn virus plasmids were extracted from the positive colonies to be used for viral packaging. The recombinant adenovirus vectors pAdxsi-GFP-IKK2dn and Lipofectamine2000 liposomes were co-transfected into 293T cells of human embryonic kidney cell line by liposome method. Observe the signs of cytotoxicity and pack it up, collect it, freeze and thaw it, amplify it, and then identify the virus species and centrifuge it. The adenovirus was purified and detected by dialysate method. The adenovirus particles were transfected into the lactation cells hela and verified its expression in the cells. Results: 1) the recombinant Shuttle-GFP-CMV(-)TEMP-IKK2dn shuttle vector plasmid of 7.4kbp was obtained. The result of pAdxsi-GFP-IKK2dn sequencing of adenovirus vector was correct. 3) finally, the titer of adenovirus vector was 2x1011 PFU / ml. 4) RT-PCR was used to verify the successful transfection of adenovirus particles into mammalian cells, which could be used for further experimental study. Conclusion: the recombinant shuttle vector plasmid of Shuttle-GFP-CMV(-)TEMP-IKK2dn was successfully constructed. The recombinant adenovirus vector pAdxsi-GFP-IKK2dn.f3) the recombinant adenovirus vectors pAdxsi-GFP-IKK2dn and Lipofectamine2000 liposomes were successfully packaged into high-titer adenovirus particles. In order to further study the induction of immune tolerance in allogeneic organ transplantation with IKK2dn gene, Hela was stained with lactation cells. Protection of grafts provides theoretical and experimental basis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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