表达NT4-SAC-HA2-TAT融合肽的重组腺伴随病毒载体的构建及鉴定
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摘要
研究背景及目的
前列腺癌是男性泌尿生殖系统常见恶性肿瘤,近年来发病率迅速增加。雄激素阻断疗法是前列腺癌各个阶段最主要的治疗手段之一。但是,多数前列腺癌在平均12～18个月后逐渐对雄激素阻断疗法失去反应,转变为雄激素非依赖性前列腺癌,预后差。前列腺凋亡反应基因-4(prostate apoptosis response-4gene,par-4)是近来发现的一个促凋亡基因,其核心区凋亡肽(SAC)不需考虑癌细胞是否存在对Par-4敏感或抵抗,对激素依赖性和非激素依赖性前列腺癌细胞都能产生凋亡诱导效应。我们构建了SAC表达载体,同时引入了信号肽神经营养因子-4(neurotrohphin-4,NT_4)及穿膜肽TAT,并进一步构建分泌NT_4-SAC-HA_2-TAT融合肽的重组腺伴随病毒,为探讨Par-4核心结构域SAC作为目的基因对前列腺癌的治疗作用奠定了基础。
方法
1.设计合成SAC的正向和反向引物,应用互为引物模板法合成具有NaeⅠ和KpnⅠ酶识别位点的SAC cDNA片段。将该片段克隆到pGEM-T easy中,转化细菌筛选阳性克隆,酶切鉴定,序列测定分析。
2.NaeⅠ和KpnⅠ酶切pGEM-T-SAC,将所得SAC和已有HA_2-TAT片段克隆到具有相应酶切位点的pBV220/NT_4质粒,得到pBV220-NT_4-SAC-HA_2-TAT重组质粒。PCR扩增以EcoRⅠ、HindⅢ酶切上述质粒获取的NT_4-SAC-HA_2-TATcDNA片段,并将其克隆到pGEM-T easy中,转化细菌筛选阳性克隆,酶切鉴定,序列测定分析。
3.将NT_4-SAC-HA_2-TAT融合基因片段插入具有相应酶切位点的pSSCMV病毒载体质粒,得到重组腺伴随病毒载体质粒。
4.将已构建的重组腺伴随病毒载体质粒、腺病毒辅助质粒PFG140和包装质粒pAAV/Ad三质粒磷酸钙共沉淀法转染293细胞系,通过同源重组获得NT_4-SAC-HA_2-TAT重组腺伴随病毒载体,收集病毒,斑点杂交(Dot blot)法测定病毒滴度。
结果
1.SAC基因经DNA测序与Genebank序列一致。
2.NT_4-SAC-HA_2-TAT融合基因经琼脂糖凝胶电泳鉴定正确。
3.酶切鉴定证实已将NT_4-SAC-HA_2-TAT融合基因成功转至PSSCMV载体中;斑点杂交测定重组腺伴随病毒的滴度约为3.42×10~(10) PFU/ml～3.42×10~(11)PFU/ml。
结论
1.成功克隆Par-4核心结构域SAC基因。
2.成功构建具有穿膜功能的“NT_4-SAC-HA_2-TA”融合基因。
3.成功构建PSSCMV/NT_4-SAC-HA_2-TA重组腺伴随病毒载体,并成功包装较高浓度的重组病毒。
Backgroud and Aims
Prostate cancer is a common cancer of male genitourinary system.Its morbility has been increasing quickly in recent years.Anti-androgen therapy is the most important means of treatment in all stages of prostate cancer.However,the majority of androgen-dependent prostate cancer can change to androgen-independent prostate cancer at an average of 12～18months after the Anti-androgen therapy.The androgen-independent prostate cancer has a poor prognosis.Prostate apoptosis response gene-4 is a recently discovered pro-apoptotic gene.Its core area of apoptosis peptide can induce both androgen-dependent and androgen-independent prostate cancer to apoptosis.We construct expression vector of SAC,which includes a signal peptide Neurotrophin-4(neurotrohphin-4,NT4) and penetrating peptide TAT.We further build the recombinant adeno-associated virus which secrets NT4-SAC-HA2-TAT fusion peptide.These experiment lay the foundation for the therapeutic effect of the Par-4 core domain SAC as a target gene for prostate cancer.
Methods
1.The forward and reverse primers of SAC were designed and synthesized.By means of asymmetrical primer/template,the fragment encoding SAC was gained, which included NaeI and KpnI restriction enzyme sites.Then the synthesized fragment was cloned into vector pGEM-T easy.The positive clone was identified by restriction enzymes,and then the cloned fragment was sequenced by dideoxy-mediated chain-termination method.
2.After pGEM-T-SAC was digested by NaeI and KpnI,the SAC/NaeI,KpnI and HA_2-TAT/KpnI,XhoI were cloned into recombinant vector pBV220-NT_4/NaeI,SalI and the recombinant plasmid pBV220-NT_4-SAC-HA_2-TAT was obtained.When the recombinant plasmid was digested with EcoRⅠand HindⅢ,the fragment NT_4-SAC-HA_2-TAT we got was amplified by PCR.Then the fragment was cloned into vector pGEM-T easy and the recombinant plasmid pGEM-T-NT_4-SAC-HA_2-TAT was obtained.The positive clone was identified by restriction enzymes,and then the cloned fragment was sequenced by dideoxy-mediated chain-termination method.
3.The fusion gene NT_4-SAC-HA_2-TAT we got was inserted into the vector plasmid pSSCMV and the vecctor of NT_4-SAC-HA_2-TAT recombinant AAV was constructed.
4.The recombinant AAV viral sock was packaged.Renal embryo 293 cells were co-transfected with the rAAV vector of plasmid pSSCMV/ NT_4-SAC-HA_2-TAT,packaging plasmid pAAV/Ad and helper adenovirus plasmid pFG140.The recombinant adeno-associated virus was produced by homologous recombination of above 3 plasmids in renal embryo 293 cells and its titer was measured by quantitative dot blot hybridization.
Results
1.By DNA sequencing,we found that we obtained the sequence of SAC which included NaeI and KpnI restriction enzyme sites by pcr.Its sequence was consistent with that of gene bank.
2.After analyzing the ORF of the cloned NT_4-SAC-HA_2-TAT cDNA by the DNASIS,we found that amino acids encoded by the cloned NT_4-SAC-HA_2-TAT cDNA were identical to the published results.
3.The vecctor of NT_4-SAC-HA_2-TAT recombinant AAV was succssfully constructed.High titer of recombinant adeno-associated virus was obtained by homologous recombination in renal embryo 293 cells.
Conclusion
1.The sequence of SAC which included NaeI and KpnI restriction enzyme sites was successfully obtained by pcr.
2.The NT4-SAC-HA2-TAT fusion gene was successfully constructed.
3.The recombinant adeno-associated virus vector encoding gene NT_4-SAC-HA_2-TAT was successfully constructed and high titer of recombinant adeno-associated virus was obtained.
前列腺癌是男性泌尿生殖系统常见恶性肿瘤,近年来发病率迅速增加。雄激素阻断疗法是前列腺癌各个阶段最主要的治疗手段之一。但是,多数前列腺癌在平均12～18个月后逐渐对雄激素阻断疗法失去反应,转变为雄激素非依赖性前列腺癌,预后差。前列腺凋亡反应基因-4(prostate apoptosis response-4gene,par-4)是近来发现的一个促凋亡基因,其核心区凋亡肽(SAC)不需考虑癌细胞是否存在对Par-4敏感或抵抗,对激素依赖性和非激素依赖性前列腺癌细胞都能产生凋亡诱导效应。我们构建了SAC表达载体,同时引入了信号肽神经营养因子-4(neurotrohphin-4,NT_4)及穿膜肽TAT,并进一步构建分泌NT_4-SAC-HA_2-TAT融合肽的重组腺伴随病毒,为探讨Par-4核心结构域SAC作为目的基因对前列腺癌的治疗作用奠定了基础。
方法
1.设计合成SAC的正向和反向引物,应用互为引物模板法合成具有NaeⅠ和KpnⅠ酶识别位点的SAC cDNA片段。将该片段克隆到pGEM-T easy中,转化细菌筛选阳性克隆,酶切鉴定,序列测定分析。
2.NaeⅠ和KpnⅠ酶切pGEM-T-SAC,将所得SAC和已有HA_2-TAT片段克隆到具有相应酶切位点的pBV220/NT_4质粒,得到pBV220-NT_4-SAC-HA_2-TAT重组质粒。PCR扩增以EcoRⅠ、HindⅢ酶切上述质粒获取的NT_4-SAC-HA_2-TATcDNA片段,并将其克隆到pGEM-T easy中,转化细菌筛选阳性克隆,酶切鉴定,序列测定分析。
3.将NT_4-SAC-HA_2-TAT融合基因片段插入具有相应酶切位点的pSSCMV病毒载体质粒,得到重组腺伴随病毒载体质粒。
4.将已构建的重组腺伴随病毒载体质粒、腺病毒辅助质粒PFG140和包装质粒pAAV/Ad三质粒磷酸钙共沉淀法转染293细胞系,通过同源重组获得NT_4-SAC-HA_2-TAT重组腺伴随病毒载体,收集病毒,斑点杂交(Dot blot)法测定病毒滴度。
结果
1.SAC基因经DNA测序与Genebank序列一致。
2.NT_4-SAC-HA_2-TAT融合基因经琼脂糖凝胶电泳鉴定正确。
3.酶切鉴定证实已将NT_4-SAC-HA_2-TAT融合基因成功转至PSSCMV载体中;斑点杂交测定重组腺伴随病毒的滴度约为3.42×10~(10) PFU/ml～3.42×10~(11)PFU/ml。
结论
1.成功克隆Par-4核心结构域SAC基因。
2.成功构建具有穿膜功能的“NT_4-SAC-HA_2-TA”融合基因。
3.成功构建PSSCMV/NT_4-SAC-HA_2-TA重组腺伴随病毒载体,并成功包装较高浓度的重组病毒。
Backgroud and Aims
Prostate cancer is a common cancer of male genitourinary system.Its morbility has been increasing quickly in recent years.Anti-androgen therapy is the most important means of treatment in all stages of prostate cancer.However,the majority of androgen-dependent prostate cancer can change to androgen-independent prostate cancer at an average of 12～18months after the Anti-androgen therapy.The androgen-independent prostate cancer has a poor prognosis.Prostate apoptosis response gene-4 is a recently discovered pro-apoptotic gene.Its core area of apoptosis peptide can induce both androgen-dependent and androgen-independent prostate cancer to apoptosis.We construct expression vector of SAC,which includes a signal peptide Neurotrophin-4(neurotrohphin-4,NT4) and penetrating peptide TAT.We further build the recombinant adeno-associated virus which secrets NT4-SAC-HA2-TAT fusion peptide.These experiment lay the foundation for the therapeutic effect of the Par-4 core domain SAC as a target gene for prostate cancer.
Methods
1.The forward and reverse primers of SAC were designed and synthesized.By means of asymmetrical primer/template,the fragment encoding SAC was gained, which included NaeI and KpnI restriction enzyme sites.Then the synthesized fragment was cloned into vector pGEM-T easy.The positive clone was identified by restriction enzymes,and then the cloned fragment was sequenced by dideoxy-mediated chain-termination method.
2.After pGEM-T-SAC was digested by NaeI and KpnI,the SAC/NaeI,KpnI and HA_2-TAT/KpnI,XhoI were cloned into recombinant vector pBV220-NT_4/NaeI,SalI and the recombinant plasmid pBV220-NT_4-SAC-HA_2-TAT was obtained.When the recombinant plasmid was digested with EcoRⅠand HindⅢ,the fragment NT_4-SAC-HA_2-TAT we got was amplified by PCR.Then the fragment was cloned into vector pGEM-T easy and the recombinant plasmid pGEM-T-NT_4-SAC-HA_2-TAT was obtained.The positive clone was identified by restriction enzymes,and then the cloned fragment was sequenced by dideoxy-mediated chain-termination method.
3.The fusion gene NT_4-SAC-HA_2-TAT we got was inserted into the vector plasmid pSSCMV and the vecctor of NT_4-SAC-HA_2-TAT recombinant AAV was constructed.
4.The recombinant AAV viral sock was packaged.Renal embryo 293 cells were co-transfected with the rAAV vector of plasmid pSSCMV/ NT_4-SAC-HA_2-TAT,packaging plasmid pAAV/Ad and helper adenovirus plasmid pFG140.The recombinant adeno-associated virus was produced by homologous recombination of above 3 plasmids in renal embryo 293 cells and its titer was measured by quantitative dot blot hybridization.
Results
1.By DNA sequencing,we found that we obtained the sequence of SAC which included NaeI and KpnI restriction enzyme sites by pcr.Its sequence was consistent with that of gene bank.
2.After analyzing the ORF of the cloned NT_4-SAC-HA_2-TAT cDNA by the DNASIS,we found that amino acids encoded by the cloned NT_4-SAC-HA_2-TAT cDNA were identical to the published results.
3.The vecctor of NT_4-SAC-HA_2-TAT recombinant AAV was succssfully constructed.High titer of recombinant adeno-associated virus was obtained by homologous recombination in renal embryo 293 cells.
Conclusion
1.The sequence of SAC which included NaeI and KpnI restriction enzyme sites was successfully obtained by pcr.
2.The NT4-SAC-HA2-TAT fusion gene was successfully constructed.
3.The recombinant adeno-associated virus vector encoding gene NT_4-SAC-HA_2-TAT was successfully constructed and high titer of recombinant adeno-associated virus was obtained.
引文
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