HIV-Ⅰ TAT和PEA-Ⅱ转膜效率的研究
摘要
凋亡素(Apoptin)是鸡贫血病毒(CAV)的VP3蛋白,由121个氨基酸组成,多年来对Apoptin的研究证明它能选择性地诱导肿瘤细胞凋亡,而且在肿瘤的治疗中凋亡素是一个独立的因子,不受p53基因突变和Bcl-2基因的影响。这些特性预示凋亡蛋白有可能成为一种治疗因子选择性地诱导肿瘤细胞凋亡。但是凋亡素的作用靶点位于细胞核内,必须进入细胞核内才能发挥生物活性,所以寻找高效、安全的导向分子和转膜系统是应用影响凋亡素治疗效果的关键。
黄体生成激素释放激素(luteinizing hormone releasing hormone,LHRH)是下丘脑分泌的一种调节性腺功能的激素,研究表明LHRH受体在卵巢癌、子宫内膜癌等细胞膜上呈特征性分布,即在肿瘤细胞表面有过量表达,所以可利用LHRH为靶向载体将治疗药物定向带到肿瘤细胞,达到将药物富集于肿瘤组织目的。
近十年来,陆续发现了一些具有蛋白质转导结构域(protein transductiondomain,PTD)的蛋白质或多肽,它们可以携带多种物质,包括亲水性蛋白、多肽、DNA甚至颗粒性物质等进行细胞间/内的传输,并且不受细胞类型的限制,如TAT(11肽、13肽)、Antp(16肽)、VP22(34肽)、Transportan(28肽)、MAP(18肽)和PEA-Ⅱ(66肽)。
为了寻找高效的转膜系统,提高凋亡素重组蛋白内化效率,本研究选用肿瘤细胞导向因子LHRH为导向分子,目前研究热点的TAT和PEA-Ⅱ分别作为转膜系统,凋亡素蛋白作为效应部分,通过PCR重叠延伸技术将Apoptin基因与肿瘤细胞导向因子LHRH基因分别与HIV-1 TAT和PEA-Ⅱ转膜系统基因重组,分别获得导向部位(LHRH)-转膜系统(TAT和PEA-Ⅱ)-凋亡蛋白两种融合蛋白基因,并通过原核表达系统实现了两种凋亡素重组融合蛋白基因LHRH-TAT-Apoptin(LTA)和LHRH-PEA-Apoptin(LPA)的表达和纯化。两种重组融合蛋白LTA和LPA分别对Heta细胞的凋亡作用显示,以HIV-1 TAT为转膜系统的凋亡素重组融合蛋白组LTA作用Hela细胞后细胞凋亡率高于LPA重组融合蛋白组,说明TAT的转膜效率高于PEA-Ⅱ。同时为了进一步检测TAT和PEA-Ⅱ的转膜效率,本研究还构建了以荧光蛋白(GFP)为报告基因的凋亡素重组融合蛋白基因LHRH-TAT-GFP-Apoptin(LTGA)和LHRH-PEA-GFP-Apoptin(LPGA)原核表达载体,经IPTG诱导表达并纯化。通过两种重组融合蛋白LTGA和LPGA分别作用Hela细胞后荧光强度显示,以TAT为转膜系统重组融合蛋白组LTGA作用Hela细胞后的绿色荧光明显强于PEA-Ⅱ重组融合蛋白组LPGA,进一步说明TAT的转膜效率高于PEA-Ⅱ。
通过上述实验,一致证实TAT的转膜效率高于PEA-Ⅱ,确定了以TAT作为转膜系统可提高重组蛋白的内化效率,增强凋亡素诱导肿瘤细胞凋亡的能力,为进一步应用凋亡素重组融合蛋白导向治疗肿瘤奠定了基础。
Recently, the regulating roles in cells by products of virus gene CAV is a new virus which can induce the apoptosis of tumor cells. The CAV derived protein VP3(Apoptin) which consists of 121 amino acids has been shown to induce apoptosis in various tumor cells, which can specifically induce apoptosis of tumor cells but does not affect normal cells or diploid cells. Experiments showed that apoptin did not induce apoptosis of normal cells. Tumor-targeting therapy will be satisfactory for enhancing therapeutic effects of anti-tumor medicines. However, the clinical application for apoptin is limited by the fact that natural apoptin can not enter cellular interior where it exerts apoptosis-inducing activity. So look for efficiently, safety of protein molecule ducting technique is appliance affect an apoptosis vegetable to treat effect of key agent.
Protein transduction technology becomes a newly emerging macromolecule transduction strategy in recent years: Direct delivery therapeutic active biomacromolecule into cells to show biological effect making use of the proteins and peptides with protein transduction domain. These CPPs such as TAT,Antp,VP22,Transportan,MAP9and PEA- II. Many kinds of substances can be delivered by CPP, there are proteins, DNA, antibodies, imaging, agent, toxin, manometer chemicals and liposome, etc.
For looking for to efficiently turn membrane system, raise an apoptosis plain and heavy histone internalization an efficiency, this research carries on genetic engineering reforming to the apoptosis plain gene, the gene difference of the direction cytokine LHRH and the gene of PEA II and TAT membrane system a gene reforming and carry on turn a membrane efficiency of comparison,Therefore if the genes encoding TAT (PEA) could be integrated into the gene encoding apoptin, the ability of apoptin to promote the apoptosis of tumor cells would be enhanced greatly.
1 Expression and purification of LTA and LPA fusion gene in E.coli and trarsmembrane efficiency of examine Applied layer after layer extension PCR technique the apoptosis plain gene and LHRH gene distinguish and TAT gene and PEA-II gene the reforming , in acquiring the fusion gene recombinant plasmid of the apoptosis pMD 18- T-LHRH-TAT(PEA )- Apoptin, The LTA and LPA genes of Fusion Proteins amplified by LA PCR and was ligated with an expression vector pET-28a to construct the prokaryotic expression and plasmid pET-28a-LHRH-TAT (PEA ) -apoptin. The LTA recombinant plasmids were transformed into host strain E.coli BL21(DE3) and induced to express LTA by IPTG. The specific protein expressed (about 19.3KDa) was detected by 15%SDS-PAGE. The protein was expressed at high level, amounting to 12.6% of the total bacterial protein as confirmed by thin-layer scanning. The LPA recombinant plasmids were transformed into host strain E.coli BL21(DE3) and induced to express LPA by IPTG. The specific protein expressed (about 27.1KDa) was detected by 15%SDS-PAGE. The protein was expressed at high level, amounting to 11.4% of the total bacterial protein as confirmed by thin-layer scanning.mainly in the form of inclusion bodies. Pass the Ni ion pillar affinity chromatography brightening LTA and the LPA apoptosis plain and heavy histone,Western blot showed that the antiserum raised against the recombinant protein could react to the protein expressed specifically.The flow cytometry examines the apoptosis plain and heavy histone's apoptosis action for swollen oncocyte, the LTA apoptosis plain and heavy Hela cell of the histone act on 72 hs after, the apoptosis rate is 31.84%;LPA apoptosis plain and heavy Hela cell of the histone act on the empress of the 72 h, the apoptosis rate is 16.24% and the matched control is 1.83%.Show as a result that compared with the matched control, the LTA fusion albumen is obviously strong to the apoptosis action of swollen oncocyte to blend albumen to the apoptosis action of swollen oncocyte at the LTA, the apoptosis rate which explains the turn of heavy histone membrane efficiency to the swollen oncocyte has influence and confirmed the turn of TAT membrane efficiency the Gao is at the PEA II .
2 Expression and purification of GFP fusion gene in E.coli and trars efficiency of examine Appliance layer after layer extension PCR technique distinguish the GFP gene and the LTA gene and LPA gene reorganize, acquire LTGA and LPGA to reorganize fusion albumen gene, in acquiring the fusion gene recombinant plasmid of the apoptosis albumen pMD 18-T-GFP, successfully set up reforming expression the plasmid as pET-28a-LHRH-TAT(PEA II)-GFP-Apoptin. The LTGA recombinant plasmids were transformed into host strain E.coli BL21(DE3) and induced to express by IPTG. The specific protein expressed (about 46.3KDa) was detected by 12%SDS-PAGE. The LPGA recombinant plasmids were transformed into host strain E.coli BL21(DE3) and induced to express by IPTG. The specific protein expressed (about 42.7KDa) was detected by 12%SDS-PAGE. With expectation the molecular weight agree with. Induce the condition as IPTG eventually the concentration 1 mmol/L, the heavy histone expresses quantity while inducing time as 4 h tallest. Use to dissolve enzyme legal system to have inclusion body albumen, express that successful brightening LTGA and LPGA reorganize fusion albumen as a result. After LTGA and the LPGA heavy act on Hela of the difference cell 72 h, the green fluorescence intensity observed TAT heavy LTGA is obviously strong in under the equal condition the heavy LPGA of PEA II , but the matched control Hela have no green fluorescence in the cell. The heavy which explains band TAT turns a membrane efficiency the heavy of the PEA. II.
This research passes apoptosis rate and the examination of fluorescence intensity, consistent certificate the TAT have to higherly turn a membrane efficiency, make sure to efficiently turn membrane system, excellent turn the design project that the apoptosis plain reforming blends albumen, raise its intemalization efficiency, strengthenned the apoptosis vegetable's concentration in intracellulare enzyme, raised an apoptosis efficiency, lay basal for the direction treatment which studies tumor further.
黄体生成激素释放激素(luteinizing hormone releasing hormone,LHRH)是下丘脑分泌的一种调节性腺功能的激素,研究表明LHRH受体在卵巢癌、子宫内膜癌等细胞膜上呈特征性分布,即在肿瘤细胞表面有过量表达,所以可利用LHRH为靶向载体将治疗药物定向带到肿瘤细胞,达到将药物富集于肿瘤组织目的。
近十年来,陆续发现了一些具有蛋白质转导结构域(protein transductiondomain,PTD)的蛋白质或多肽,它们可以携带多种物质,包括亲水性蛋白、多肽、DNA甚至颗粒性物质等进行细胞间/内的传输,并且不受细胞类型的限制,如TAT(11肽、13肽)、Antp(16肽)、VP22(34肽)、Transportan(28肽)、MAP(18肽)和PEA-Ⅱ(66肽)。
为了寻找高效的转膜系统,提高凋亡素重组蛋白内化效率,本研究选用肿瘤细胞导向因子LHRH为导向分子,目前研究热点的TAT和PEA-Ⅱ分别作为转膜系统,凋亡素蛋白作为效应部分,通过PCR重叠延伸技术将Apoptin基因与肿瘤细胞导向因子LHRH基因分别与HIV-1 TAT和PEA-Ⅱ转膜系统基因重组,分别获得导向部位(LHRH)-转膜系统(TAT和PEA-Ⅱ)-凋亡蛋白两种融合蛋白基因,并通过原核表达系统实现了两种凋亡素重组融合蛋白基因LHRH-TAT-Apoptin(LTA)和LHRH-PEA-Apoptin(LPA)的表达和纯化。两种重组融合蛋白LTA和LPA分别对Heta细胞的凋亡作用显示,以HIV-1 TAT为转膜系统的凋亡素重组融合蛋白组LTA作用Hela细胞后细胞凋亡率高于LPA重组融合蛋白组,说明TAT的转膜效率高于PEA-Ⅱ。同时为了进一步检测TAT和PEA-Ⅱ的转膜效率,本研究还构建了以荧光蛋白(GFP)为报告基因的凋亡素重组融合蛋白基因LHRH-TAT-GFP-Apoptin(LTGA)和LHRH-PEA-GFP-Apoptin(LPGA)原核表达载体,经IPTG诱导表达并纯化。通过两种重组融合蛋白LTGA和LPGA分别作用Hela细胞后荧光强度显示,以TAT为转膜系统重组融合蛋白组LTGA作用Hela细胞后的绿色荧光明显强于PEA-Ⅱ重组融合蛋白组LPGA,进一步说明TAT的转膜效率高于PEA-Ⅱ。
通过上述实验,一致证实TAT的转膜效率高于PEA-Ⅱ,确定了以TAT作为转膜系统可提高重组蛋白的内化效率,增强凋亡素诱导肿瘤细胞凋亡的能力,为进一步应用凋亡素重组融合蛋白导向治疗肿瘤奠定了基础。
Recently, the regulating roles in cells by products of virus gene CAV is a new virus which can induce the apoptosis of tumor cells. The CAV derived protein VP3(Apoptin) which consists of 121 amino acids has been shown to induce apoptosis in various tumor cells, which can specifically induce apoptosis of tumor cells but does not affect normal cells or diploid cells. Experiments showed that apoptin did not induce apoptosis of normal cells. Tumor-targeting therapy will be satisfactory for enhancing therapeutic effects of anti-tumor medicines. However, the clinical application for apoptin is limited by the fact that natural apoptin can not enter cellular interior where it exerts apoptosis-inducing activity. So look for efficiently, safety of protein molecule ducting technique is appliance affect an apoptosis vegetable to treat effect of key agent.
Protein transduction technology becomes a newly emerging macromolecule transduction strategy in recent years: Direct delivery therapeutic active biomacromolecule into cells to show biological effect making use of the proteins and peptides with protein transduction domain. These CPPs such as TAT,Antp,VP22,Transportan,MAP9and PEA- II. Many kinds of substances can be delivered by CPP, there are proteins, DNA, antibodies, imaging, agent, toxin, manometer chemicals and liposome, etc.
For looking for to efficiently turn membrane system, raise an apoptosis plain and heavy histone internalization an efficiency, this research carries on genetic engineering reforming to the apoptosis plain gene, the gene difference of the direction cytokine LHRH and the gene of PEA II and TAT membrane system a gene reforming and carry on turn a membrane efficiency of comparison,Therefore if the genes encoding TAT (PEA) could be integrated into the gene encoding apoptin, the ability of apoptin to promote the apoptosis of tumor cells would be enhanced greatly.
1 Expression and purification of LTA and LPA fusion gene in E.coli and trarsmembrane efficiency of examine Applied layer after layer extension PCR technique the apoptosis plain gene and LHRH gene distinguish and TAT gene and PEA-II gene the reforming , in acquiring the fusion gene recombinant plasmid of the apoptosis pMD 18- T-LHRH-TAT(PEA )- Apoptin, The LTA and LPA genes of Fusion Proteins amplified by LA PCR and was ligated with an expression vector pET-28a to construct the prokaryotic expression and plasmid pET-28a-LHRH-TAT (PEA ) -apoptin. The LTA recombinant plasmids were transformed into host strain E.coli BL21(DE3) and induced to express LTA by IPTG. The specific protein expressed (about 19.3KDa) was detected by 15%SDS-PAGE. The protein was expressed at high level, amounting to 12.6% of the total bacterial protein as confirmed by thin-layer scanning. The LPA recombinant plasmids were transformed into host strain E.coli BL21(DE3) and induced to express LPA by IPTG. The specific protein expressed (about 27.1KDa) was detected by 15%SDS-PAGE. The protein was expressed at high level, amounting to 11.4% of the total bacterial protein as confirmed by thin-layer scanning.mainly in the form of inclusion bodies. Pass the Ni ion pillar affinity chromatography brightening LTA and the LPA apoptosis plain and heavy histone,Western blot showed that the antiserum raised against the recombinant protein could react to the protein expressed specifically.The flow cytometry examines the apoptosis plain and heavy histone's apoptosis action for swollen oncocyte, the LTA apoptosis plain and heavy Hela cell of the histone act on 72 hs after, the apoptosis rate is 31.84%;LPA apoptosis plain and heavy Hela cell of the histone act on the empress of the 72 h, the apoptosis rate is 16.24% and the matched control is 1.83%.Show as a result that compared with the matched control, the LTA fusion albumen is obviously strong to the apoptosis action of swollen oncocyte to blend albumen to the apoptosis action of swollen oncocyte at the LTA, the apoptosis rate which explains the turn of heavy histone membrane efficiency to the swollen oncocyte has influence and confirmed the turn of TAT membrane efficiency the Gao is at the PEA II .
2 Expression and purification of GFP fusion gene in E.coli and trars efficiency of examine Appliance layer after layer extension PCR technique distinguish the GFP gene and the LTA gene and LPA gene reorganize, acquire LTGA and LPGA to reorganize fusion albumen gene, in acquiring the fusion gene recombinant plasmid of the apoptosis albumen pMD 18-T-GFP, successfully set up reforming expression the plasmid as pET-28a-LHRH-TAT(PEA II)-GFP-Apoptin. The LTGA recombinant plasmids were transformed into host strain E.coli BL21(DE3) and induced to express by IPTG. The specific protein expressed (about 46.3KDa) was detected by 12%SDS-PAGE. The LPGA recombinant plasmids were transformed into host strain E.coli BL21(DE3) and induced to express by IPTG. The specific protein expressed (about 42.7KDa) was detected by 12%SDS-PAGE. With expectation the molecular weight agree with. Induce the condition as IPTG eventually the concentration 1 mmol/L, the heavy histone expresses quantity while inducing time as 4 h tallest. Use to dissolve enzyme legal system to have inclusion body albumen, express that successful brightening LTGA and LPGA reorganize fusion albumen as a result. After LTGA and the LPGA heavy act on Hela of the difference cell 72 h, the green fluorescence intensity observed TAT heavy LTGA is obviously strong in under the equal condition the heavy LPGA of PEA II , but the matched control Hela have no green fluorescence in the cell. The heavy which explains band TAT turns a membrane efficiency the heavy of the PEA. II.
This research passes apoptosis rate and the examination of fluorescence intensity, consistent certificate the TAT have to higherly turn a membrane efficiency, make sure to efficiently turn membrane system, excellent turn the design project that the apoptosis plain reforming blends albumen, raise its intemalization efficiency, strengthenned the apoptosis vegetable's concentration in intracellulare enzyme, raised an apoptosis efficiency, lay basal for the direction treatment which studies tumor further.
引文
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[48] Miyazaki M, Schally AV, NagyA, et al. Targeted cytotoxic analog of luteinizing hormone-releasing hormone AN-207 inhibits growth of OV-1063 human epithelial ovarian cancers in nude mice.Am J Obstet Gynecol,1999.180:1095-1103
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[50]Shanna A,Sharma MS,De Padua M,et al.Synchronous endometrial carcinoma and a macroprolactinoma:exploring a causal relationship.Oncology.2007.72(1-2):139-142
[51]Schally AV,Comaru-schally AM,Nagy A,et al.Hypothalamic hormones and cancer.Frontiers in Neuroendocrinology,2001.22:248-291
[52]Lai CH,Huang HJ.The role of hormones for the treatment of endometrial hyperplasia and endometrial cancer.Curt Opin Obstet Gynecol.2006 Feb.18(1):9-34
[53]Grundker C,Volker P,Griesinger F,et al.Antitumor effects ofcytotoxic LHRH analog AN-207 on human endometrial and ovarian cancers xenografted into nude mice.American Journal of Obstetrics and Gynecology 2002.187:528-537
[54]Schally AV,Comaru-Schally AM.Hypothalamic and other peptide hormones.In:Bast RC,Kufe DW,Pollock RE,Weichselbaum RR,Holland JF,Frei Ⅲ E,Eds.Cancer Medicine,5#ed.Lewiston,NY:Decker.2000.715-729
[55]寇冰.VP3/Apoptin融合蛋白诱导肿瘤细胞凋亡及人肝癌裸鼠移植瘤生长抑制的研究:[博士学位论文].武汉:华中科技大学,2006
[56]Thompson C B.Apoptosis in the pathogenesis and treatment of disease Science,1995.267(10):1456-1460
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[59] Liu X, Elojeimy S, El-Zawahry AM, et al. Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer. Mol Ther. 2006 Nov. 14(5): 637-646
[60] Related Articles, LinksTavassoli Apoptin: specific killer of tumor cells? Apoptosis. 2005 Aug. 10(4): 717-724
[61] Kozak M. A analysis of 5'-noncoding sequence from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1993.15: 8125-8148
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[63] Koch G, van-Roozelaar DJ, Verschueren CA, et al. Im munogenic and protective properties of chicken anaemia virus proteins expressed by baclovirus. Vaccine, 1995. 13(8): 763-770
[64] He X, Zhang Q, Liu Y, et al. Apoptin Induces Chromatin Condensation in Normal Cells.Virus Genes. 2005 Aug. 31(1): 49-55
[65] Leliveld SR, Zbang YH, Rolm JL, et al. Apoptin induces tumor-specific apoplosis as a globular multimer. Biol Chem, 2003. 278(9): 42-51
[66] Leliveld SR, Dame RT, Mommaas VIA, et al. Apoptin protein multimers form distinct higher-order nucleoprotein complexes with DNA. Nucleic Acid Res, 2003.31(1): 805-813
[67] Zhuang SM, Landegent JL, Verschueren CA, et al. Apoptin, a protein encoded by chicken anemia virus induces cell death in various human hematological malignant cells in vitro. Leukemia, 1995. 9: 118-120
[68]Olijslagers SJ,Zhang YH,Backendorf C,et al.Additive cytotoxic effect of apoptin and chemotherapeutic agents paclitaxel and etoposide on human tumour cells.Basic Clin Pharmacol Toxicol.2007 Feb.100(2):127-131
[69]Noteborn MH,Zhann YH,van der AJ.Apoptin specifically causes apoptosis in tumor cells and after UV-treatment in untransformed cells from cancer-prone individuals a review.Mutat Res,1998.400:447-452
[70]Danen-van,Oorschot AAM,van der EAJ.Bcl2 stimulates apoptin-induced apoptosis.Adv Exp Med Biol,1999.457:245-249
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[72]Rohn JL,Zhang YI I,Aalbers RLJ,et al.A rumor-specific kinase activity regulates the viral death protein Apoptin.Biol Chcm,2002.277(50):820-827
[73]Pietersen AM.Precliniral studies with Apoptin.PhD thesis,Erasmus University,Rotterdam,The Netherlands 2003
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[76]李士泽.凋亡蛋白融合基因的克隆、表达及其对肿瘤细胞凋亡的影响:[博士学位论文].吉林:军需大学,2004
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[78]ChalfieM Tu Y,Euchirchen G,Prashert D C,et al.Green fluo rescent protein as a marker for gene expression.Science,1994,263(11):802-805
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[81]Liu LB,Li WM,Zou P,et al.Depressing the immune escape of acute myelomonocytic leukemia via an anti-Fas ribozyme.Zhongguo Shi Yan Xue Ye Xue Za Zhi.2006 Oct.14(5):862-866
[82]郝军元.凋亡素重组蛋白导向治疗肿瘤的研究:[博士学位论文].吉林:吉林大学,2006
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[48] Miyazaki M, Schally AV, NagyA, et al. Targeted cytotoxic analog of luteinizing hormone-releasing hormone AN-207 inhibits growth of OV-1063 human epithelial ovarian cancers in nude mice.Am J Obstet Gynecol,1999.180:1095-1103
[49]Miyazaki M,Nagy A,Schally AV,et al.Growth ingibition of human ovarian cancers by cytotoxic analogues of luteinizing hormone=releasing hormone.ournal of the National Cancer Institute 1997.89:1803-1809
[50]Shanna A,Sharma MS,De Padua M,et al.Synchronous endometrial carcinoma and a macroprolactinoma:exploring a causal relationship.Oncology.2007.72(1-2):139-142
[51]Schally AV,Comaru-schally AM,Nagy A,et al.Hypothalamic hormones and cancer.Frontiers in Neuroendocrinology,2001.22:248-291
[52]Lai CH,Huang HJ.The role of hormones for the treatment of endometrial hyperplasia and endometrial cancer.Curt Opin Obstet Gynecol.2006 Feb.18(1):9-34
[53]Grundker C,Volker P,Griesinger F,et al.Antitumor effects ofcytotoxic LHRH analog AN-207 on human endometrial and ovarian cancers xenografted into nude mice.American Journal of Obstetrics and Gynecology 2002.187:528-537
[54]Schally AV,Comaru-Schally AM.Hypothalamic and other peptide hormones.In:Bast RC,Kufe DW,Pollock RE,Weichselbaum RR,Holland JF,Frei Ⅲ E,Eds.Cancer Medicine,5#ed.Lewiston,NY:Decker.2000.715-729
[55]寇冰.VP3/Apoptin融合蛋白诱导肿瘤细胞凋亡及人肝癌裸鼠移植瘤生长抑制的研究:[博士学位论文].武汉:华中科技大学,2006
[56]Thompson C B.Apoptosis in the pathogenesis and treatment of disease Science,1995.267(10):1456-1460
[57]Liu SS,Ben SB,Zhao HL.Construction of apoptin gene delivery system and its effect on apoptosis of A375 cells.Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi.2008Feb.24(2):133-135
[58] Jeurissen SHM, et al. Chicken anemia virus cause apoptosis of thymocytes after in vivo infection and of cell lines after in vitro infection. Virol, 1992. 66: 7383-7386
[59] Liu X, Elojeimy S, El-Zawahry AM, et al. Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer. Mol Ther. 2006 Nov. 14(5): 637-646
[60] Related Articles, LinksTavassoli Apoptin: specific killer of tumor cells? Apoptosis. 2005 Aug. 10(4): 717-724
[61] Kozak M. A analysis of 5'-noncoding sequence from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1993.15: 8125-8148
[62] Heckl S, Regenbogen M, Sturzu A, et al. Value of apoptin's 40-amino-acid C-terminal fragment for the differentiation between human tumor and non-tumor cells. Apoptosis. 2008 Apr. 13(4): 495-508
[63] Koch G, van-Roozelaar DJ, Verschueren CA, et al. Im munogenic and protective properties of chicken anaemia virus proteins expressed by baclovirus. Vaccine, 1995. 13(8): 763-770
[64] He X, Zhang Q, Liu Y, et al. Apoptin Induces Chromatin Condensation in Normal Cells.Virus Genes. 2005 Aug. 31(1): 49-55
[65] Leliveld SR, Zbang YH, Rolm JL, et al. Apoptin induces tumor-specific apoplosis as a globular multimer. Biol Chem, 2003. 278(9): 42-51
[66] Leliveld SR, Dame RT, Mommaas VIA, et al. Apoptin protein multimers form distinct higher-order nucleoprotein complexes with DNA. Nucleic Acid Res, 2003.31(1): 805-813
[67] Zhuang SM, Landegent JL, Verschueren CA, et al. Apoptin, a protein encoded by chicken anemia virus induces cell death in various human hematological malignant cells in vitro. Leukemia, 1995. 9: 118-120
[68]Olijslagers SJ,Zhang YH,Backendorf C,et al.Additive cytotoxic effect of apoptin and chemotherapeutic agents paclitaxel and etoposide on human tumour cells.Basic Clin Pharmacol Toxicol.2007 Feb.100(2):127-131
[69]Noteborn MH,Zhann YH,van der AJ.Apoptin specifically causes apoptosis in tumor cells and after UV-treatment in untransformed cells from cancer-prone individuals a review.Mutat Res,1998.400:447-452
[70]Danen-van,Oorschot AAM,van der EAJ.Bcl2 stimulates apoptin-induced apoptosis.Adv Exp Med Biol,1999.457:245-249
[71]Darien-van,Fisher DF,Grimhergen JM,et al.Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells.Proc Natl Acad Sci USA,1997.94(5):843-847
[72]Rohn JL,Zhang YI I,Aalbers RLJ,et al.A rumor-specific kinase activity regulates the viral death protein Apoptin.Biol Chcm,2002.277(50):820-827
[73]Pietersen AM.Precliniral studies with Apoptin.PhD thesis,Erasmus University,Rotterdam,The Netherlands 2003
[74]Pietersen AM,Rademalcer HJ,et al.Specific tumor-cell killing with adenovirus vocter containing the Apoptin gene.Gene Ther,1999.6:882-892
[75]Prasher D C,Eckenrode V K,WardW W,et al.Primary structure of the A equerea victoria green fiuo rescent protein Gene.1992.111:229-233
[76]李士泽.凋亡蛋白融合基因的克隆、表达及其对肿瘤细胞凋亡的影响:[博士学位论文].吉林:军需大学,2004
[77]Cody C W,Prasher D C,rendergast F G,et al.Chemical structure of the hexapeptide chromophore of the A equorea green-fluo rescent protein.Biochemistry,1993.32:1212-1220
[78]ChalfieM Tu Y,Euchirchen G,Prashert D C,et al.Green fluo rescent protein as a marker for gene expression.Science,1994,263(11):802-805
[80]Wang,et al.Implications for bcd mRNA localization from spatial distribution of in drosophila oogendsis.Nature,1994.369:400- 403
[81]Liu LB,Li WM,Zou P,et al.Depressing the immune escape of acute myelomonocytic leukemia via an anti-Fas ribozyme.Zhongguo Shi Yan Xue Ye Xue Za Zhi.2006 Oct.14(5):862-866
[82]郝军元.凋亡素重组蛋白导向治疗肿瘤的研究:[博士学位论文].吉林:吉林大学,2006