携带EphrinA1-caspase-3基因的重组腺病毒载体的构建和包装
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摘要
目的:构建携带ephrinA1-caspase-3基因的重组腺病毒载体,通过包装、扩增、鉴定,为后续开展目的基因靶向治疗乳腺癌研究奠定基础。方法:以pMD18T-Casp3、pMD18T-EphrinA1为模板扩增caspase-3、EphrinA1基因,以pET28a为载体,通过双酶切、连接、转化、测序鉴定,构建pET28a-EphrinA1-Casp3。然后以质粒pEC3.1(+)为载体,获得pEC3.1-EphrinA1-Casp3。采用LR体外同源重组,构建pAd-EphrinA1-Casp3,以HEK293包装和放大培养。结果:成功将PCR扩增的EphrinA1胞外端基因和人活性型caspase-3基因定向克隆入质粒pET28a,并将EphrinA1-caspase-3融合基因转移到穿梭质粒pEC3.1(+),获得pEC3.1-EphrinA1-Casp3载体;经过体外同源重组,成功构建携带EphrinA1-caspase-3基因的重组腺病毒载体pAd-EphrinA1-Casp3;并在HEK 293细胞中完成包装和扩增;获得重组腺病毒rAd-EphrinA1-Casp3。结论:利用GatewayTM技术成功构建重组腺病毒载体pAd-EphrinA1-Casp3,经HEK293细胞包装,获得重组腺病毒rAdEphrinA1-Casp3。
Objective:To construct the recombinant adenovirus vector carrying the EphrinA1-caspase-3 gene,and to lay agood experimental foundation for the further research on the targeting treatment of breast cancer with the desired gene after packaging and amplification.Methods:The caspase-3 gene and EphrinA1 gene were amplified by PCR with the clone vector pMD18 T-Casp3 containing the human active caspase-3 gene and pMD18 T-EphrinA1 as the templates,respectively.The PCR product of these genes and the plasmid pET28 awere digested with double restriction enzymes,and then were collected,connected,and transformed into E.coli DH5α competent cells.The positive clones with correct gene sequencing were named pET28 a-EphrinA1-Casp3.Then the pET28 a-EphrinA1-Casp3 and the shuttle vector pEC3.1(+)were connected after double enzyme digestion,and the pEC3.1-EphrinA1-Casp3 was obtained after transformation and sequencing analysis.And then the expression cassette of EphrinA1-Casp3 was transferred to pAd/pl-DEST adenoviral vector in vitro with LR homologous recombinant reaction and the recombinant adenovirus plasmid pAd-EphrinA1-Casp3 was produced.In the end the pAdEphrinA1-Casp3 was linearized and transfected into HEK293 cells for packing and amplification,and the recombinant adenovirus rAd-EphrinA1-Casp3 was identified by PCR and obtained.Results:The PCR product of the human active caspase-3 gene and that of the EphrinA1 gene were cloned into the adenovirus vector pET28 ain the correct direction successfully,and the fused gene EphrinA1-caspase-3 was transferred to the adenovirus shuttle vector pEC3.1(+)to acquire recombinant plasmid pEC3.1-EphrinA1-Casp3.The recombinant adenovirus vector pAd-EphrinA1-Casp3 containing EphrinA1-Casp3 gene was constructed successfully by the application of LR homologous recombinant reactionin vitro.And after the transfection of pAdEphrinA1-Casp3 into HEK293 cells for packing and amplification,the recombinant adenovirus rAd-EphrinA1-Casp3 was obtained.Conclusion:The recombinant adenovirus vector pAd-EphrinA1-Casp3 expressing EphrinA1-Casp3 gene can be constructed successfully with GatewayTM technology,and the recombinant adenovirus rAd-EphrinA1-Casp3 is prepared through packing and amplification in HEK293 cells.
Objective:To construct the recombinant adenovirus vector carrying the EphrinA1-caspase-3 gene,and to lay agood experimental foundation for the further research on the targeting treatment of breast cancer with the desired gene after packaging and amplification.Methods:The caspase-3 gene and EphrinA1 gene were amplified by PCR with the clone vector pMD18 T-Casp3 containing the human active caspase-3 gene and pMD18 T-EphrinA1 as the templates,respectively.The PCR product of these genes and the plasmid pET28 awere digested with double restriction enzymes,and then were collected,connected,and transformed into E.coli DH5α competent cells.The positive clones with correct gene sequencing were named pET28 a-EphrinA1-Casp3.Then the pET28 a-EphrinA1-Casp3 and the shuttle vector pEC3.1(+)were connected after double enzyme digestion,and the pEC3.1-EphrinA1-Casp3 was obtained after transformation and sequencing analysis.And then the expression cassette of EphrinA1-Casp3 was transferred to pAd/pl-DEST adenoviral vector in vitro with LR homologous recombinant reaction and the recombinant adenovirus plasmid pAd-EphrinA1-Casp3 was produced.In the end the pAdEphrinA1-Casp3 was linearized and transfected into HEK293 cells for packing and amplification,and the recombinant adenovirus rAd-EphrinA1-Casp3 was identified by PCR and obtained.Results:The PCR product of the human active caspase-3 gene and that of the EphrinA1 gene were cloned into the adenovirus vector pET28 ain the correct direction successfully,and the fused gene EphrinA1-caspase-3 was transferred to the adenovirus shuttle vector pEC3.1(+)to acquire recombinant plasmid pEC3.1-EphrinA1-Casp3.The recombinant adenovirus vector pAd-EphrinA1-Casp3 containing EphrinA1-Casp3 gene was constructed successfully by the application of LR homologous recombinant reactionin vitro.And after the transfection of pAdEphrinA1-Casp3 into HEK293 cells for packing and amplification,the recombinant adenovirus rAd-EphrinA1-Casp3 was obtained.Conclusion:The recombinant adenovirus vector pAd-EphrinA1-Casp3 expressing EphrinA1-Casp3 gene can be constructed successfully with GatewayTM technology,and the recombinant adenovirus rAd-EphrinA1-Casp3 is prepared through packing and amplification in HEK293 cells.
引文
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[14]阙文忠,陈君敏.利用Gateway技术构建重组腺病毒pAd-NK4[J].中国药理学通报,2011,27(4):462-466.
[2]Shariat S F,Desai S,Song W,et al.Adenovirus-mediated transfer of inducible caspases:A novel“death switch”gene therapeutic approach to prostate cancer[J].Cancer Res,2001,61(6):2562-2571.
[3]Pasquale E B.Eph receptors and ephrins in cancer:bidirectional signalling and beyond[J].Nat Rev Cancer,2010,10(3):165-180.
[4]Liu Y,Yu C,Qiu Y,et al.Down regulation of EphA2expression suppresses the growth and metastasis in squamous-cell carcinoma of the head and neck in vitro and invivo[J].J Cancer Res Clin Oncol,2012,138(2):195-202.
[5]Cui X D,Lcc M J,Yu G R,et al.EFNAl ligand and its receptor EphA2:potential biomarkers for hepatocellular carcinoma[J].Int J Cancer,2010,126(4):940-949.
[6]Udayakumar D,Zhang G,Ji Z,et al.EphA2is a critical oncogene in melanoma[J].Oncogene,2011,30(50):4921-4929.
[7]张本斯,卞思源,潘云,等.乳腺癌EphA2和EphrinA1的表达及与临床病理因素的关系[J].中国临床解剖学杂志,2017,35(1):43-47.
[8]Du A,Zhao B,Yin D,et al.Safrole oxide induces apoptosis by activating caspase-3,-8,and-9in A549human lung cancer cells[J].Bioorg Med Chem Lett,2006,l6(1):81-83.
[9]Srinivasula S M,Ahmad M,MacFarlane M,et al.Generation of constitutively active recombinant caspase-3and-6rearrangement of their subtmits[J].J Boil Chem,1998,273(17):10107-10111.
[10]Partridge K A,Oreffo R O.Gene delivery in bone tissue engineering:progress and prospects using viral and nonviral strategies[J].Tissue Eng,2004,10(1-2):295-307.
[11]Douglas J T.Adenoviral vectors for gene therapy[J].Mol Biotechnol,2007,36(1):71-80.
[12]Russell W C.Update on adenovirus and its vectors[J].Gene,2007,81(7):2573-2604.
[13]He T C,Zhou S,Da C L,et al.A simplified system for generating recombinant adenoviruses[J].Proc Natl Acad Sci U S A,1998,95(5):2509-2514.
[14]阙文忠,陈君敏.利用Gateway技术构建重组腺病毒pAd-NK4[J].中国药理学通报,2011,27(4):462-466.