携带p53基因和Flt3L配体基因的腺病毒载体的构建及鉴定
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摘要
目的：以MUC1为启动子构建携带p53/F1t3L融合基因的腺病毒载体,表达p53基因和Flt3L基因,发挥p53抑癌作用和树突状细胞的免疫治疗、诱导乳腺癌细胞的凋亡的作用,为进一步探讨重组腺病毒载体对乳腺癌的治疗作用提供条件。
方法：通过人工合成MUC1启动子、p53基因和Flt3L配体基因,并在序列两端加入相应的酶切位点,通过酶切、连接等方法将其分别克隆至pUC19载体中,合成pUC19-MUC1、pUC19-p53和pUC19-Flt3L,将反应液全量转化至感受态细胞并进行质粒DNA提取,经DNA序列测定证实目的基因合成正确。利用相应的限制性内切酶将上述三个质粒进行重组,合成并提取质粒pUC19-MUC1-p53和pUC19-MUC1-Flt3L.通过PCR扩增p53基因和Flt3L配体基因,应用linker序列将两个基因连在一起构成融合基因,亚克隆至pUC19-MUC1质粒中,经双酶切、琼脂糖凝胶电泳鉴定并回收目的片段,对pUC19一MUC1-p53-linker-Flt3L进行测序分析。利用多克隆位点,应用限制性内切酶进行双酶切,回收目的DNA片段并进行磷酸化及末端补平,将目的基因分别插入到在被线性化的pAxcwit2腺病毒载体中,构建成pAx-MUC 1一p53.pAx-MUC 1一Flt3L. pAx-MUC 1一p53一linker-Flt3L,对三个重组质粒进行DNA测序分析及酶切鉴定,证实插入的外源基因与已知gene bank上相应的基因序列一致,构建应用MUC1作为启动子携带p53/Flt3L融合基因的腺病毒载体成功。
结果：人工合成的MUC1启动子、p53基因和Flt3L配体基因序列经DNA序列测定证实与已知gene bank给出的基因序列完全一致,重组质粒pAx-MUC 1-p53.pAx-MUC 1-Flt3L.pAx-MUC1-p53-linker-Flt3L经DNA序列测序、酶切及琼脂糖凝胶电泳鉴定,结果表明重组腺病毒载体中外源基因序列与已知序列一致,15%琼脂糖凝胶电泳释放条带长度与已知基因片段长度一致,证实合成基因正确,构建腺病毒载体成功。
结论：
1.通过基因合成技术成功合成MUC1启动子、p53基因和Flt3L配体基因,与gene bank已知序列完全一致,为基因重组创造了必要的条件。
2.成功构建应用MUC1作为启动子的腺病毒载体,采用MUC1作为启动子,进一步增强后期实验腺病毒载体对于乳腺癌细胞的靶向性治疗。
3.成功构建了应用MUC1作为启动子携带p53/Flt3L融合基因的腺病毒载体。
Objective:To construct adenoviral vector containing MUC1 promoter and the fusion gene of p53/Flt3L,to express the gene of p53/Flt3L.Give full play to the role of p53 tumor suppressor and Immune therapy of dendritic cells,to induce the apoptosis of breast cancer cells.To further explore the conditions the adenovirus vector provide for the treatment of breast cancer.
Methods:Artificial synthetic MUC1 promoter,p53 gene and Flt3 ligand gene,and adding the appropriate restriction sites in the two sides of the sequence,and useing the method of restriction enzymolysis,connection to clone them into pUC19 vector,and pUC19-MUC1,pUC19-p53 and pUC19-Flt3L were obtained,the whole volume of the reaction solution was transformed into the competent cells and extract plasmid DNA and the DNA sequencing was confirmed to be correct.restructure the above three plasmids using the appropriate restriction enzyme,synthesis and extraction the plasmids of pUC19-MUC1-p53 and pUC19-MUC1-Flt3L.p53 gene and Flt3 ligand were amplified by PCR,the fusion gene was constructed by using a linker sequences,the fusion gene was inserted the into plasmid of pUC19-MUC1 plasmid,the plasmid was verified by double digesti-on, agarose gel electrophoresis and the fragment was recovered.The pUC19-MUC1-p53-linker-Flt3L were sequenced.Using the multiple cloning sites, double restriction enzyme to digestion,the DNA fragment of purpose were recovered,phosphorylated and end up flated.The objective gene were inserted into the line of the pAxcwit2 of adenovirus vector,the recombinant plasmid of pAx-MUC1-p53,pAx-MUC1-Flt3L,pAx-MUC1-p53-linker-Flt3L were successfully constructed.Confirm the DNA sequencing and restriction analysis of the three recombinant plasmid and find that the inserted foreign gene were correspond with corresponding gene sequences of gene bank.It demonstrated that recombinant adenoviral vector carrying MUC1 promoter and fusion of p53/Flt3L was successfully constructed.
Results:The synthetic MUC1 promoter,p53 gene and Flt3 ligand sequence were confirmed by DNA sequencing that the inserted foreign gene were correspond with corresponding gene sequences of gene bank.The recombinant plasmids of pAx-MUC1-p53,pAx-MUC1-Flt3L,pAx-MUC1-p53-linker-Flt3L were confirmed by the method DNA sequencing,restriction enzyme digestion and agarose gel electrophoresis,the results show that the exogenous gene sequences of recombinant adenovirus vector were consistent with the known sequences.The band length of 15% agarose gel electrophoresis released were consistent with the known gene fragment.It confirmed the synthesis gene were proper correct,adenovirus vectors were successfully constructed.
Conclusion:
1. The adenovirus vector MUC1 promoter and fusion gene of p53/Flt3L constructed by gene synthesis technology were consistent with the known sequences,that create the necessary conditions for recombinanting DNA.
2. Successfully applicat MUC1 as a promoter constructed adenovirus vector,using MUC1 as a promoter to further enhance the targeting of adenovirus vector for the treatment of breast cancer cells in the later experiments.
3. Adenovirus vector using MUC1 as promoter and bringing p53/Flt3L fusion gene was successfully constructed.
方法：通过人工合成MUC1启动子、p53基因和Flt3L配体基因,并在序列两端加入相应的酶切位点,通过酶切、连接等方法将其分别克隆至pUC19载体中,合成pUC19-MUC1、pUC19-p53和pUC19-Flt3L,将反应液全量转化至感受态细胞并进行质粒DNA提取,经DNA序列测定证实目的基因合成正确。利用相应的限制性内切酶将上述三个质粒进行重组,合成并提取质粒pUC19-MUC1-p53和pUC19-MUC1-Flt3L.通过PCR扩增p53基因和Flt3L配体基因,应用linker序列将两个基因连在一起构成融合基因,亚克隆至pUC19-MUC1质粒中,经双酶切、琼脂糖凝胶电泳鉴定并回收目的片段,对pUC19一MUC1-p53-linker-Flt3L进行测序分析。利用多克隆位点,应用限制性内切酶进行双酶切,回收目的DNA片段并进行磷酸化及末端补平,将目的基因分别插入到在被线性化的pAxcwit2腺病毒载体中,构建成pAx-MUC 1一p53.pAx-MUC 1一Flt3L. pAx-MUC 1一p53一linker-Flt3L,对三个重组质粒进行DNA测序分析及酶切鉴定,证实插入的外源基因与已知gene bank上相应的基因序列一致,构建应用MUC1作为启动子携带p53/Flt3L融合基因的腺病毒载体成功。
结果：人工合成的MUC1启动子、p53基因和Flt3L配体基因序列经DNA序列测定证实与已知gene bank给出的基因序列完全一致,重组质粒pAx-MUC 1-p53.pAx-MUC 1-Flt3L.pAx-MUC1-p53-linker-Flt3L经DNA序列测序、酶切及琼脂糖凝胶电泳鉴定,结果表明重组腺病毒载体中外源基因序列与已知序列一致,15%琼脂糖凝胶电泳释放条带长度与已知基因片段长度一致,证实合成基因正确,构建腺病毒载体成功。
结论：
1.通过基因合成技术成功合成MUC1启动子、p53基因和Flt3L配体基因,与gene bank已知序列完全一致,为基因重组创造了必要的条件。
2.成功构建应用MUC1作为启动子的腺病毒载体,采用MUC1作为启动子,进一步增强后期实验腺病毒载体对于乳腺癌细胞的靶向性治疗。
3.成功构建了应用MUC1作为启动子携带p53/Flt3L融合基因的腺病毒载体。
Objective:To construct adenoviral vector containing MUC1 promoter and the fusion gene of p53/Flt3L,to express the gene of p53/Flt3L.Give full play to the role of p53 tumor suppressor and Immune therapy of dendritic cells,to induce the apoptosis of breast cancer cells.To further explore the conditions the adenovirus vector provide for the treatment of breast cancer.
Methods:Artificial synthetic MUC1 promoter,p53 gene and Flt3 ligand gene,and adding the appropriate restriction sites in the two sides of the sequence,and useing the method of restriction enzymolysis,connection to clone them into pUC19 vector,and pUC19-MUC1,pUC19-p53 and pUC19-Flt3L were obtained,the whole volume of the reaction solution was transformed into the competent cells and extract plasmid DNA and the DNA sequencing was confirmed to be correct.restructure the above three plasmids using the appropriate restriction enzyme,synthesis and extraction the plasmids of pUC19-MUC1-p53 and pUC19-MUC1-Flt3L.p53 gene and Flt3 ligand were amplified by PCR,the fusion gene was constructed by using a linker sequences,the fusion gene was inserted the into plasmid of pUC19-MUC1 plasmid,the plasmid was verified by double digesti-on, agarose gel electrophoresis and the fragment was recovered.The pUC19-MUC1-p53-linker-Flt3L were sequenced.Using the multiple cloning sites, double restriction enzyme to digestion,the DNA fragment of purpose were recovered,phosphorylated and end up flated.The objective gene were inserted into the line of the pAxcwit2 of adenovirus vector,the recombinant plasmid of pAx-MUC1-p53,pAx-MUC1-Flt3L,pAx-MUC1-p53-linker-Flt3L were successfully constructed.Confirm the DNA sequencing and restriction analysis of the three recombinant plasmid and find that the inserted foreign gene were correspond with corresponding gene sequences of gene bank.It demonstrated that recombinant adenoviral vector carrying MUC1 promoter and fusion of p53/Flt3L was successfully constructed.
Results:The synthetic MUC1 promoter,p53 gene and Flt3 ligand sequence were confirmed by DNA sequencing that the inserted foreign gene were correspond with corresponding gene sequences of gene bank.The recombinant plasmids of pAx-MUC1-p53,pAx-MUC1-Flt3L,pAx-MUC1-p53-linker-Flt3L were confirmed by the method DNA sequencing,restriction enzyme digestion and agarose gel electrophoresis,the results show that the exogenous gene sequences of recombinant adenovirus vector were consistent with the known sequences.The band length of 15% agarose gel electrophoresis released were consistent with the known gene fragment.It confirmed the synthesis gene were proper correct,adenovirus vectors were successfully constructed.
Conclusion:
1. The adenovirus vector MUC1 promoter and fusion gene of p53/Flt3L constructed by gene synthesis technology were consistent with the known sequences,that create the necessary conditions for recombinanting DNA.
2. Successfully applicat MUC1 as a promoter constructed adenovirus vector,using MUC1 as a promoter to further enhance the targeting of adenovirus vector for the treatment of breast cancer cells in the later experiments.
3. Adenovirus vector using MUC1 as promoter and bringing p53/Flt3L fusion gene was successfully constructed.
引文
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