新型的以组蛋白H1～0为骨架的靶向于EGF受体的基因导入系统的构建
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摘要
本文构建了一种以组蛋白H10为骨架，靶向于表皮生长因子（EGF）受体的新型基因导入系统。其创新性在于，把基因导入系统中的各种功能成分组建成为融合基因，在大肠杆菌中表达，是化学合成基因导入系统的延续，属相关领域的首次尝试与报道。
在以往的研究中，我们构建了多聚赖氨酸（polylysine）为骨架、靶向于EGF受体的基因导入系统。GE7-polylysine和HA20-polylysine是系统的两个组成成分。GE7是一种化学合成的16肽，靶向于EGF受体。HA20来源于流感病毒血凝素蛋白，是内吞小体释放寡肽。功能肽GE7、HA20分别通过化学交联与polylysine连接。在体内、体外导入实验中，这种polylysine为骨架的基因导入系统能够靶向性地将DNA导入EGF受体高表达的肿瘤细胞。但化学交联很难确定与polylysine连接的寡肽数目并且花费昂贵，限制了进一步的应用。基因工程方法能解决这些问题，但在大肠杆菌体内不可能表达polylysine，因此我们设计了组蛋白H10为骨架的基因导入系统。我们总共构建了九种融合蛋白，它们分别为GE7-histone-HA20, GE7-histone97-193-HA20, HA20-histone-GE7, Histone-GE7, Histone-HA20, Histone97-193-GE7, Histone97-193-HA20， Histone，Histone97-193。Histone和Histone97-193两种蛋白作为阴性对照。
组蛋白H10和组蛋白H1097-193通过PCR从人胎盘cDNA文库中获得。与组蛋白H10或组蛋白H1097-193连接的GE7和HA20是从本人构建的表达序列GE7-protamine和HA20-protamine中PCR获得。通过一系列重叠的引物，数轮PCR"搭桥"反应最终获得九个融合蛋白的表达序列。所有的表达序列均克隆在pT7450原核表达载体中。
融合蛋白的表达宿主菌是HMS174（DE3），表达产物通过SP Sepharose和Sephadex G-50纯化。
所有的融合蛋白与质粒DNA混合后，都能阻滞DNA在1%琼脂糖凝胶中的迁移，电泳结果表明融合蛋白具有结合DNA的能力。DNA与蛋白结合的最佳质量比为1:3。我们采用了经免疫组化鉴定表达EGF受体的SKOV3裸鼠皮下移植瘤、BEL-7402细胞株、BEL-7402裸鼠皮下移植瘤作为靶细胞、组织以检测融合蛋白的生物学活性。U2OS细胞表面未检测出EGF受体的表达，作为导入实验的阴性对照。体外导入实验表明，只有2种蛋白组合（GE7-histone-HA20/Histone-GE7, Histone-GE7/Histone- HA20）能将(-gal基因导入表达EGF受体的细胞，其他组合或单独的蛋白均不能将DNA靶向性地导入EGF受体高表达的细胞。体内导入实验表明，未连接GE7或HA20的组蛋白不能将DNA靶向性地导入细胞，单独Histone-HA20也是同样的结果；单独的Histone-GE7，GE7-histone-HA20，HA20-histone-GE7组的肿瘤中仅检测出少量(-gal基因的表达；将Histone-GE7和Histone-HA20等量混合后，(-gal基因的表达有所增加；GE7-histone-HA20和Histone-GE7等量混合后，SKOV3和BEL-7402肿瘤中可检测到大量(-gal基因的表达。体外导入筛选实验表明，Histone97-193为骨架的基因导入系统活性不如Histone全长。GE7-histone-HA20/Histone-GE7蛋白组合是经体内外导入实验筛选获得的最佳基因导入系统。
我们同时研究了GE7-histone-HA20/Histone-GE7蛋白组合介导的(-gal基因在肿瘤组织中的表达随时间推移的变化：在皮下瘤周注射后12小时，就有(-gal基因的表达，24小时表达达到高峰，48小时开始下降，96小时、7天、10天表达继续下降，2周后肿瘤组织内仍有少量(-gal基因表达。肿瘤组织中导入的(-gal基因的表达水平还随复合体剂量的增加而上升。含DNA 1(g的复合体是皮下瘤周注射的最佳剂量。
这一设计思路将基因导入系统中各种功能成分连接后通过基因工程方法表达，属首次尝试与报道，因此仅仅是一个起点。由于给药后，复合体颗粒要历经细胞内外的重重障碍，才能到达靶细胞核，在整个过程中，有众多干扰因素阻碍着复合体的导入进程，增加复合体颗粒的稳定性，避免被降解是该研究今后的发展方向。通过聚乙二醇化可使DNA与融合蛋白形成的复合体稳定性增加。这是在颗粒进入靶细胞前，维持稳定不被降解的对策。通过对复合体颗粒的修饰，完善基因导入系统，以排除细胞内外的干扰，将为今后的临床基因治疗奠定基础。
The construction of a novel gene delivery system based on Histone H10 targeting epidermal growth factor receptor (EGF R) and its expression in E.coli was described. It is an extension of the research on chemically synthetic composite polypeptide gene delivery system in our lab. So far, it is the first trial in gene delivery system research. The biological activity of the expressed polypeptide was studied.
In previous research, we have constructed a polylysine-based gene delivery system targeting EGF R. It was composed of GE7-polylysine and HA20-polylysine. GE7 was a synthetic peptide (16 amino acids) targeting EGF R. HA20 was derived from influenza virus hemagglutinin, acting as an endosome-releasing oligopeptide. The functional oligopeptides of GE7 and HA20 have been covalently conjugated to polylysine. This polylysine-based gene delivery system was capable of delivering DNA of exogenous gene to tumor cells over-expressing EGF R both in vitro and in vivo. But the uncertainty of cross-link made it difficult to control the number of oligopeptide linked to polylysine and the homogeneity of the final products.
To solve these problems, a series of Histone H10-based fused genes which contain Histone H10, GE7 or/and HA20 were constructed and expressed in E.coli. We have managed to express 9 fused proteins: GE7-histone-HA20, GE7-histone97-193-HA20, HA20-histone-GE7, Histone-GE7, Histone-HA20, Histone97-193-GE7, Histone97-193-HA20, Histone and Histone97-193, among which the last two were taken as negative control.
In the process of construction, Histone H10 and Histone H1097-193 were isolated from a placenta cDNA library by using designed primers and PCR. The GE7 and HA20 linked to
Histone H10 and Histone H1097-193 were prepared by PCR from previous constructs of GE7-protamine and HA20-protamine. The DNA sequences of the above 9 fused genes were constructed by several cycles of PCR with overlapped primers. Then all the sequences were cloned into pT7450 prokaryotic expression vector. The nine fused proteins have been expressed in E.coli HMS174 (DE3), and purified by SP Sepharose and Sephadex G-50.
After mixed with plasmid DNA, all of these proteins were capable of retarding DNA mobility in 1% agarose gel, indicating the binding ability of the fused proteins to DNA. The optimal ratio of DNA to fused protein was 1:3 by weight.
We have detected EGF R expression in SKOV3 transplanted tumor tissue, BEL-7402 cells and its transplanted tumor by immunohistochemical analysis. They were taken as target cells to verify the delivery capability of fused proteins. U2OS cells, on which no EGF R was detected, were used as a negative control.
To test the biological activity of this gene delivery system, we have conducted experiments both in vitro and in vivo. In vitro, only two combinations of proteins (GE7-histone-HA20/Histone-GE7, Histone-GE7/Histone-HA20) could deliver (-galactosidase gene to EGF R expressing cells. Other combination and single fused proteins failed to deliver gene targeting EGF R. In vivo, we have found that Histone without GE7 or HA20 was incapable of delivering (-galactosidase gene targeting EGF R expressing cells. Neither was the case of Histone-HA20. By using Histone-GE7, GE7-histone-HA20, and HA20-histone-GE7, slight extent of (-galactosidase activity could be detected. When we used DNA complexed with Histone-GE7/Histone-HA20, slightly more (-galactosidase activity was detected. However, by using a 1:1 mixture of GE7-histone-HA20 and Histone-GE7 as delivery system, much more (-galactosidase activity was detected both in SKOV3 and BEL-7402 tumor cells in vivo. So far, the combined fused protein GE7-histone-HA20 and Histone-GE7 seems to be the optimal gene delivery system.
We have also examined the time course of (-gal gene expression delivered into the tumor cells by GE7-histone-HA20/Histone-GE7. We found that (-gal gene began to express from 12 hours after peritumoral injection. At 24 hour, the expression reached the peak, and it began to decline at 48 hour. Then
在以往的研究中，我们构建了多聚赖氨酸（polylysine）为骨架、靶向于EGF受体的基因导入系统。GE7-polylysine和HA20-polylysine是系统的两个组成成分。GE7是一种化学合成的16肽，靶向于EGF受体。HA20来源于流感病毒血凝素蛋白，是内吞小体释放寡肽。功能肽GE7、HA20分别通过化学交联与polylysine连接。在体内、体外导入实验中，这种polylysine为骨架的基因导入系统能够靶向性地将DNA导入EGF受体高表达的肿瘤细胞。但化学交联很难确定与polylysine连接的寡肽数目并且花费昂贵，限制了进一步的应用。基因工程方法能解决这些问题，但在大肠杆菌体内不可能表达polylysine，因此我们设计了组蛋白H10为骨架的基因导入系统。我们总共构建了九种融合蛋白，它们分别为GE7-histone-HA20, GE7-histone97-193-HA20, HA20-histone-GE7, Histone-GE7, Histone-HA20, Histone97-193-GE7, Histone97-193-HA20， Histone，Histone97-193。Histone和Histone97-193两种蛋白作为阴性对照。
组蛋白H10和组蛋白H1097-193通过PCR从人胎盘cDNA文库中获得。与组蛋白H10或组蛋白H1097-193连接的GE7和HA20是从本人构建的表达序列GE7-protamine和HA20-protamine中PCR获得。通过一系列重叠的引物，数轮PCR"搭桥"反应最终获得九个融合蛋白的表达序列。所有的表达序列均克隆在pT7450原核表达载体中。
融合蛋白的表达宿主菌是HMS174（DE3），表达产物通过SP Sepharose和Sephadex G-50纯化。
所有的融合蛋白与质粒DNA混合后，都能阻滞DNA在1%琼脂糖凝胶中的迁移，电泳结果表明融合蛋白具有结合DNA的能力。DNA与蛋白结合的最佳质量比为1:3。我们采用了经免疫组化鉴定表达EGF受体的SKOV3裸鼠皮下移植瘤、BEL-7402细胞株、BEL-7402裸鼠皮下移植瘤作为靶细胞、组织以检测融合蛋白的生物学活性。U2OS细胞表面未检测出EGF受体的表达，作为导入实验的阴性对照。体外导入实验表明，只有2种蛋白组合（GE7-histone-HA20/Histone-GE7, Histone-GE7/Histone- HA20）能将(-gal基因导入表达EGF受体的细胞，其他组合或单独的蛋白均不能将DNA靶向性地导入EGF受体高表达的细胞。体内导入实验表明，未连接GE7或HA20的组蛋白不能将DNA靶向性地导入细胞，单独Histone-HA20也是同样的结果；单独的Histone-GE7，GE7-histone-HA20，HA20-histone-GE7组的肿瘤中仅检测出少量(-gal基因的表达；将Histone-GE7和Histone-HA20等量混合后，(-gal基因的表达有所增加；GE7-histone-HA20和Histone-GE7等量混合后，SKOV3和BEL-7402肿瘤中可检测到大量(-gal基因的表达。体外导入筛选实验表明，Histone97-193为骨架的基因导入系统活性不如Histone全长。GE7-histone-HA20/Histone-GE7蛋白组合是经体内外导入实验筛选获得的最佳基因导入系统。
我们同时研究了GE7-histone-HA20/Histone-GE7蛋白组合介导的(-gal基因在肿瘤组织中的表达随时间推移的变化：在皮下瘤周注射后12小时，就有(-gal基因的表达，24小时表达达到高峰，48小时开始下降，96小时、7天、10天表达继续下降，2周后肿瘤组织内仍有少量(-gal基因表达。肿瘤组织中导入的(-gal基因的表达水平还随复合体剂量的增加而上升。含DNA 1(g的复合体是皮下瘤周注射的最佳剂量。
这一设计思路将基因导入系统中各种功能成分连接后通过基因工程方法表达，属首次尝试与报道，因此仅仅是一个起点。由于给药后，复合体颗粒要历经细胞内外的重重障碍，才能到达靶细胞核，在整个过程中，有众多干扰因素阻碍着复合体的导入进程，增加复合体颗粒的稳定性，避免被降解是该研究今后的发展方向。通过聚乙二醇化可使DNA与融合蛋白形成的复合体稳定性增加。这是在颗粒进入靶细胞前，维持稳定不被降解的对策。通过对复合体颗粒的修饰，完善基因导入系统，以排除细胞内外的干扰，将为今后的临床基因治疗奠定基础。
The construction of a novel gene delivery system based on Histone H10 targeting epidermal growth factor receptor (EGF R) and its expression in E.coli was described. It is an extension of the research on chemically synthetic composite polypeptide gene delivery system in our lab. So far, it is the first trial in gene delivery system research. The biological activity of the expressed polypeptide was studied.
In previous research, we have constructed a polylysine-based gene delivery system targeting EGF R. It was composed of GE7-polylysine and HA20-polylysine. GE7 was a synthetic peptide (16 amino acids) targeting EGF R. HA20 was derived from influenza virus hemagglutinin, acting as an endosome-releasing oligopeptide. The functional oligopeptides of GE7 and HA20 have been covalently conjugated to polylysine. This polylysine-based gene delivery system was capable of delivering DNA of exogenous gene to tumor cells over-expressing EGF R both in vitro and in vivo. But the uncertainty of cross-link made it difficult to control the number of oligopeptide linked to polylysine and the homogeneity of the final products.
To solve these problems, a series of Histone H10-based fused genes which contain Histone H10, GE7 or/and HA20 were constructed and expressed in E.coli. We have managed to express 9 fused proteins: GE7-histone-HA20, GE7-histone97-193-HA20, HA20-histone-GE7, Histone-GE7, Histone-HA20, Histone97-193-GE7, Histone97-193-HA20, Histone and Histone97-193, among which the last two were taken as negative control.
In the process of construction, Histone H10 and Histone H1097-193 were isolated from a placenta cDNA library by using designed primers and PCR. The GE7 and HA20 linked to
Histone H10 and Histone H1097-193 were prepared by PCR from previous constructs of GE7-protamine and HA20-protamine. The DNA sequences of the above 9 fused genes were constructed by several cycles of PCR with overlapped primers. Then all the sequences were cloned into pT7450 prokaryotic expression vector. The nine fused proteins have been expressed in E.coli HMS174 (DE3), and purified by SP Sepharose and Sephadex G-50.
After mixed with plasmid DNA, all of these proteins were capable of retarding DNA mobility in 1% agarose gel, indicating the binding ability of the fused proteins to DNA. The optimal ratio of DNA to fused protein was 1:3 by weight.
We have detected EGF R expression in SKOV3 transplanted tumor tissue, BEL-7402 cells and its transplanted tumor by immunohistochemical analysis. They were taken as target cells to verify the delivery capability of fused proteins. U2OS cells, on which no EGF R was detected, were used as a negative control.
To test the biological activity of this gene delivery system, we have conducted experiments both in vitro and in vivo. In vitro, only two combinations of proteins (GE7-histone-HA20/Histone-GE7, Histone-GE7/Histone-HA20) could deliver (-galactosidase gene to EGF R expressing cells. Other combination and single fused proteins failed to deliver gene targeting EGF R. In vivo, we have found that Histone without GE7 or HA20 was incapable of delivering (-galactosidase gene targeting EGF R expressing cells. Neither was the case of Histone-HA20. By using Histone-GE7, GE7-histone-HA20, and HA20-histone-GE7, slight extent of (-galactosidase activity could be detected. When we used DNA complexed with Histone-GE7/Histone-HA20, slightly more (-galactosidase activity was detected. However, by using a 1:1 mixture of GE7-histone-HA20 and Histone-GE7 as delivery system, much more (-galactosidase activity was detected both in SKOV3 and BEL-7402 tumor cells in vivo. So far, the combined fused protein GE7-histone-HA20 and Histone-GE7 seems to be the optimal gene delivery system.
We have also examined the time course of (-gal gene expression delivered into the tumor cells by GE7-histone-HA20/Histone-GE7. We found that (-gal gene began to express from 12 hours after peritumoral injection. At 24 hour, the expression reached the peak, and it began to decline at 48 hour. Then
引文
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44. Gottschalk S, Sparrow JT, Hauer J, et al. A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells. Gene Ther 1996, 3: 448-457
45. Kichler A, Mechtler K, Behr JP, et al. The influence of membrane-active peptides and helper lipids on lipospermine/DNA complex mediated gene transfer. Bioconjugate Chem 1997, 8: 213-221
46. Wyman TB, Nicol F, Zelphati O, et al. Design, synthesis and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry 1997, 36: 3008-3017
47. Wagner E, Plank C, Zatloukal K, et al. Influenza virus hemagglutinin HA2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes; toward a synthetic virus-like gene transfer vehicle. Proc Natl Acad Sci USA 1992, 89: 7934-7938
Murata, M., Kagiwada, S., Takahashi, SH., et al. Membrane fusion induced by mutual
48. interaction of the two charge-reversed amphiphilic peptides at neutral pH. J Biol Chem 1991, 266: 14353-14358
49. 田培坤，任圣俊，任常春，等。一种新的以细胞表面受体为靶向的基因导入系统。中国科学1998, 28：554-560
50. Sambrook J., Fritsch E.F. and Maniatis T. Molecular cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989
51. Owens RJ, Young RJ. The genetic engineering of monoclonal antibody. J Immunol Methods. 1994, 168:149-165
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56. Doenecke D, Tonjes R. Differential distribution of lysine and arginine residues in the closely related histones H1 and H5. J Mol Biol 1986, 187: 461-464
57. Antoni Kozlowski J, Milton Harris. Improvements in protein PEGylation: PEGylated interferons for treatment of hepatitis C. Journal of Controlled Release 2001, 72: 217-224
58. Wolfgang Zauner, Manfred Ogris, Ernst Wagner. Polylysine-based transfection systems utilizing receptor-mediated delivery. Advanced Drug Delivery Reviews 1998, 30: 97-113
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43. Plank C, Oberhauser B, Mechtler K, et al. The influence of endosome-disruptive peptides on gene transfer using synthetic virus like gene transfer systems. J Biol Chem 1994, 269: 12918-12924
44. Gottschalk S, Sparrow JT, Hauer J, et al. A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells. Gene Ther 1996, 3: 448-457
45. Kichler A, Mechtler K, Behr JP, et al. The influence of membrane-active peptides and helper lipids on lipospermine/DNA complex mediated gene transfer. Bioconjugate Chem 1997, 8: 213-221
46. Wyman TB, Nicol F, Zelphati O, et al. Design, synthesis and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry 1997, 36: 3008-3017
47. Wagner E, Plank C, Zatloukal K, et al. Influenza virus hemagglutinin HA2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes; toward a synthetic virus-like gene transfer vehicle. Proc Natl Acad Sci USA 1992, 89: 7934-7938
Murata, M., Kagiwada, S., Takahashi, SH., et al. Membrane fusion induced by mutual
48. interaction of the two charge-reversed amphiphilic peptides at neutral pH. J Biol Chem 1991, 266: 14353-14358
49. 田培坤，任圣俊，任常春，等。一种新的以细胞表面受体为靶向的基因导入系统。中国科学1998, 28：554-560
50. Sambrook J., Fritsch E.F. and Maniatis T. Molecular cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989
51. Owens RJ, Young RJ. The genetic engineering of monoclonal antibody. J Immunol Methods. 1994, 168:149-165
52. 鄂征. 组织培养和分子细胞学技术. 北京出版社,1995
53. 蛋白质电泳实验技术。郭尧君 编著。科学出版社，1999。2 ISBN 7-03- 006899-8
54. Fritz JD, Herweijer H, Zhang G, et al. Gene transfer into mammalian cells using histone-condensed plasmid DNA. Hum Gene Ther 1996, 7: 1395-1404
55. Jian Chen, Robert J, Katherine A, et al. Galactosylated histone-mediated gene transfer and expression. Hum Gene Ther 1994, 5: 429-435
56. Doenecke D, Tonjes R. Differential distribution of lysine and arginine residues in the closely related histones H1 and H5. J Mol Biol 1986, 187: 461-464
57. Antoni Kozlowski J, Milton Harris. Improvements in protein PEGylation: PEGylated interferons for treatment of hepatitis C. Journal of Controlled Release 2001, 72: 217-224
58. Wolfgang Zauner, Manfred Ogris, Ernst Wagner. Polylysine-based transfection systems utilizing receptor-mediated delivery. Advanced Drug Delivery Reviews 1998, 30: 97-113