重组人白介素24在大肠杆菌中的高效表达及其抗肿瘤应用
发布时间：2020-10-31 16:29
P. B. Fisher等人在细胞表面受体Mda-7发现的基础上，鉴定了黑素瘤分化相关蛋白-7（MDA-7），随后MDA-7被重命名为IL-24。IL24属于IL-10细胞因子家族成员，该家族包括IL-10、IL-19、IL-20、IL-22、IL-24和IL-26。IL-24具有抑制多种人类癌细胞生长的特性，同时对正常细胞无诱导危害。IL-24具有潜在的治疗应用价值并在肿瘤细胞学领域起着重要作用。 重组人源IL-24（rhIL-24）最初是使用Escherichia coli进行生产并利用传统方法进行纯化鉴定，其发酵策略是基于LB培养基的分批培养，然后利用高压均质机来裂解细胞。在E.coli中表达真核生物蛋白会导致不溶性的包涵体（IBs）的形成。对活性治疗性蛋白生产来说，包涵体的溶解和重折叠是一个关键挑战。传统的包涵体溶解和重折叠方法纯化应用于小体积的样品，使用阴离子和阳离子交换色谱法从样品中取出杂质，从而得到纯化的产品。这种传统的rhIL-24制备方法被认为是一个耗时、费力的过程。本文设计了一种基于传统方法的高效、低成本的rhIL-24制备策略。培养基的成本和组成对于商业规模的E.coli重组蛋白生产至关重要，采用向培养基中添加酵母提取物、葡萄糖等成分提高rhIL-24的产量；同时，用乳糖替代IPTG的使用，也降低了成本和毒性。此外，我们用一种2步变性-重折叠法（2DR）替代传统的重折叠法，从而提高了rhIL-24的产量。新的制备策略使得高质量、高纯度的rhIL-24获得过程更加简单，同时，LC-MS/MS技术的运用为rhIL-24提供了精确的定性、定量信息。 一步纯化的rhIL-24被作为癌症和多药耐药肿瘤细胞的有效的治疗剂进行相关分析。rhIL-24在肝癌细胞系HepG2显示了生物活性，但是对L02细胞没有影响。膜上的P-糖蛋白（P-gp）是多药耐药的主要因素。我们研究了rhIL-24对抗阿霉素（ADM）人源乳腺癌细胞系MCF-7/ADM的影响，利用MTT法对rhIL24和ADM的细胞毒性进行了检测，P-gp的表达用显微共聚焦法和Western blot法分析评估，ADM在MCF-7/ADM细胞系中的IC50在加入rhIL-24后呈现剂量依赖性降低。在低剂量的rhIL-24（(4μPol·L-1)）的MCF-7/ADM细胞系中，ADM积累增加，而P-gp的表达降低。体外生物活性表明，rhIL-24通过激活Stat3转录因子绕过MCF-7/ADM细胞的多药耐药，rhIL-24可以作为P-糖蛋白的抑制剂，用以逆转乳腺癌细胞的阿霉素抗性。 本研究的最终目标是将rhIL-24作为可控释放药物。基于干细胞的基因治疗方案，受限于IL-24到皮下肿瘤的转运。腺病毒介导的mda-7/IL-24(Ad.mda-7)法会导致过度表达引起细胞生长停滞和凋亡。在病毒载体相比，非病毒载体系统具有毒性低，生物相容性，可控性。非病毒载体与药物的共轭聚合物可以显著增加药物的水溶性，改性的组织分布和血浆循环半衰期。蛋白质-PEG复合物已能通过增加药物的稳定性显著提高疗效。本研究开发的策略，产生一个均匀的、单体嵌合的rhIL-24和PEG鱼精蛋白复合物。PEG鱼精蛋白-IL24纳米颗粒能够确认对MCF-7／ADM细胞活性。PEG修饰的鱼精蛋白–rhIL-24纳米颗粒具有无可比拟的疗效。目前的研究对一种rhIL-24控释制剂的临床实现铺平了道路。
【学位单位】：江南大学
【学位级别】：博士
【学位年份】：2014
【中图分类】：R378.21
【文章目录】：
摘要
ABSTRACT
LIST OF ABBREVIATION
TABLE OF CONTENS
Chapter 1 Introduction and literature review
1.1 Recombinant Human Interleukin
1.1.1 Melanoma Differentiation Associated Gene-7/IL24 and melanoma
1.1.2 Apoptosis Inducing Properties of IL24
1.1.3 Antitumor Bystander Activity of IL24
1.1.4 Anti -angiogenic activity of IL24
1.1.5 Combination Therapy with IL24
1.1.6 IL24 and Mycobacterium Tuberculosis
1.2 IL24 in Cancer Therapy
1.3 Recombinant protein production
1.4 Nanotechnology
1.5 Research Objective
Chapter 2 A conventional method for fermentation and purification of Recombinant Human interleukin 24 from E. coli
2.1 Introduction
2.2 Materials and Methods
2.2.1 Fermentation Media
2.2.2 Batch Cultivation
2.3 rh-IL24 purification
2.3.1 Cell lysis and IB recovery
2.3.2 IBs washing:
2.3.3 IB solubilization and refolding
2.3.4 Anion & Cation Exchange Chromatography
2.3.5 SDS-PAGE
2.4 Results and Discussion
2.4.1 Cell lysis and IB isolation
2.4.2 IB washing
2.4.3 IB solubilization and refolding
2.4.4 Anion & Cation Exchange Chromatography
2.5 Conclusion
Chapter 3 Cost effective production of rhIL24
3.1 Introduction
3.2 Materials and methods
3.2.1 Bacterial strain and vector system
3.2.2 Induction and expression of rhIL24 in culture flasks
3.2.3 3L Fermentation of rhIL24
3.2.4 Disruption, washing and isolation of inclusion bodies （IBs）
3.2.5 Two-step denaturing and refolding of rhIL24 IBs
3.2.6 Diafiltration
3.2.7 Cation exchange chromatography
3.2.8 SDS-PAGE and western blot
3.2.9 LC-MS/MS
3.2.10 Bioactivity assay of rhIL24 in vitro
3.3 Results and discussion
3.3.1 Cloning and construction of the pET21a （+）-rhIL2417
3.3.2 Medium selection
3.3.3 Lactose induction
3.3.4 Cell lysis and inclusion body isolation
3.3.5 IB washes
3.3.6 IB solubilization and refolding
3.3.7 Cation exchange chromatography
3.3.8 Western blot analysis and LC-MS/MS
3.3.9 Biological activity assay of rhIL24
3.4 Conclusions
Chapter 4 rhIL24 reverses Adriamycin resistance in MCF-7/ADM human breast cancer cell line
4.1 Introduction
4.2. Materials and Methods
4.2.1 Bacterial Expression, Refolding, and Analysis
4.2.2 Cells and cell cultures
4.2.3 Cytotoxicity assay
4.2.4 In situ analysis of P-gp expression by confocal laser scanning microscopy
4.2.5 Western blot analysis of P-gp expression
4.3. Results
4.3.1 rhIL-24 expression and purification
4.3.2 Modulation of ADM resistance
4.3.3 P-gp expression by confocal laser scanning microscopy
4.3.4 Western blot analysis of P-gp protein expression in tumor cells
4.4. Discussion
Chapter 5 PEGylated Protamine-rhIL24 nanoparticle
5.1 Introduction
5.2 Materials and methods
5.2.1 Materials
5.2.2 Preparation of PEG-Protamine Complex
5.2.3 Preparation of PEGylated Protamine-rhIL24 Nanoparticles
5.3 Characterization
5.3.1 Gel permeation Chromatography （GPC） for PEG Protamine Complex
5.3.2 Nuclear Magnetic Resonance Spectroscopy for PEG Protamine Complex
5.3.3 Measurement of particle size, zeta potential （ζ） for PEGylated Protamine-rhIL24 Nanoparticle
5.3.4 SDS-PAGE for PEGylated Protamine-rhIL24 Nanoparticle
5.3.5 Transmission electron microscopy （TEM） for PEGylated Protamine-rhIL24 Nanoparticle
5.3.6 PEGylated Protamine/IL24 nanoparticles cytotoxicity study
5.4 Results and Discussion
5.4.1 Gel permeation Chromatography （GPC） for PEG Protamine Complex
5.4.2 Nuclear Magnetic Resonance Spectroscopy for PEG Protamine Complex
5.4.3 Measurement of particle size, zeta potential （ζ） PEGylated Protamine-rhIL24 Nanoparticle
5.4.4 SDS-PAGE of PEGylated Protamine-rhIL24 Nanoparticle
5.4.5 Transmission electron microscopy （TEM） for PEGylated Protamine-rhIL24
5.4.6 PEGylated Protamine/IL24 nanoparticles cytotoxicity study
5.5 Conclusions
Major conclusion
Recommendation for future work
Key Innovations
Acknowledgements
References
APPENDIX
【参考文献】
本文编号：2864200
【学位单位】：江南大学
【学位级别】：博士
【学位年份】：2014
【中图分类】：R378.21
【文章目录】：
摘要
ABSTRACT
LIST OF ABBREVIATION
TABLE OF CONTENS
Chapter 1 Introduction and literature review
1.1 Recombinant Human Interleukin
1.1.1 Melanoma Differentiation Associated Gene-7/IL24 and melanoma
1.1.2 Apoptosis Inducing Properties of IL24
1.1.3 Antitumor Bystander Activity of IL24
1.1.4 Anti -angiogenic activity of IL24
1.1.5 Combination Therapy with IL24
1.1.6 IL24 and Mycobacterium Tuberculosis
1.2 IL24 in Cancer Therapy
1.3 Recombinant protein production
1.4 Nanotechnology
1.5 Research Objective
Chapter 2 A conventional method for fermentation and purification of Recombinant Human interleukin 24 from E. coli
2.1 Introduction
2.2 Materials and Methods
2.2.1 Fermentation Media
2.2.2 Batch Cultivation
2.3 rh-IL24 purification
2.3.1 Cell lysis and IB recovery
2.3.2 IBs washing:
2.3.3 IB solubilization and refolding
2.3.4 Anion & Cation Exchange Chromatography
2.3.5 SDS-PAGE
2.4 Results and Discussion
2.4.1 Cell lysis and IB isolation
2.4.2 IB washing
2.4.3 IB solubilization and refolding
2.4.4 Anion & Cation Exchange Chromatography
2.5 Conclusion
Chapter 3 Cost effective production of rhIL24
3.1 Introduction
3.2 Materials and methods
3.2.1 Bacterial strain and vector system
3.2.2 Induction and expression of rhIL24 in culture flasks
3.2.3 3L Fermentation of rhIL24
3.2.4 Disruption, washing and isolation of inclusion bodies （IBs）
3.2.5 Two-step denaturing and refolding of rhIL24 IBs
3.2.6 Diafiltration
3.2.7 Cation exchange chromatography
3.2.8 SDS-PAGE and western blot
3.2.9 LC-MS/MS
3.2.10 Bioactivity assay of rhIL24 in vitro
3.3 Results and discussion
3.3.1 Cloning and construction of the pET21a （+）-rhIL2417
3.3.2 Medium selection
3.3.3 Lactose induction
3.3.4 Cell lysis and inclusion body isolation
3.3.5 IB washes
3.3.6 IB solubilization and refolding
3.3.7 Cation exchange chromatography
3.3.8 Western blot analysis and LC-MS/MS
3.3.9 Biological activity assay of rhIL24
3.4 Conclusions
Chapter 4 rhIL24 reverses Adriamycin resistance in MCF-7/ADM human breast cancer cell line
4.1 Introduction
4.2. Materials and Methods
4.2.1 Bacterial Expression, Refolding, and Analysis
4.2.2 Cells and cell cultures
4.2.3 Cytotoxicity assay
4.2.4 In situ analysis of P-gp expression by confocal laser scanning microscopy
4.2.5 Western blot analysis of P-gp expression
4.3. Results
4.3.1 rhIL-24 expression and purification
4.3.2 Modulation of ADM resistance
4.3.3 P-gp expression by confocal laser scanning microscopy
4.3.4 Western blot analysis of P-gp protein expression in tumor cells
4.4. Discussion
Chapter 5 PEGylated Protamine-rhIL24 nanoparticle
5.1 Introduction
5.2 Materials and methods
5.2.1 Materials
5.2.2 Preparation of PEG-Protamine Complex
5.2.3 Preparation of PEGylated Protamine-rhIL24 Nanoparticles
5.3 Characterization
5.3.1 Gel permeation Chromatography （GPC） for PEG Protamine Complex
5.3.2 Nuclear Magnetic Resonance Spectroscopy for PEG Protamine Complex
5.3.3 Measurement of particle size, zeta potential （ζ） for PEGylated Protamine-rhIL24 Nanoparticle
5.3.4 SDS-PAGE for PEGylated Protamine-rhIL24 Nanoparticle
5.3.5 Transmission electron microscopy （TEM） for PEGylated Protamine-rhIL24 Nanoparticle
5.3.6 PEGylated Protamine/IL24 nanoparticles cytotoxicity study
5.4 Results and Discussion
5.4.1 Gel permeation Chromatography （GPC） for PEG Protamine Complex
5.4.2 Nuclear Magnetic Resonance Spectroscopy for PEG Protamine Complex
5.4.3 Measurement of particle size, zeta potential （ζ） PEGylated Protamine-rhIL24 Nanoparticle
5.4.4 SDS-PAGE of PEGylated Protamine-rhIL24 Nanoparticle
5.4.5 Transmission electron microscopy （TEM） for PEGylated Protamine-rhIL24
5.4.6 PEGylated Protamine/IL24 nanoparticles cytotoxicity study
5.5 Conclusions
Major conclusion
Recommendation for future work
Key Innovations
Acknowledgements
References
APPENDIX
【参考文献】
相关期刊论文 前2条
1 Sunyoung Park;Soyoung Cheon;Daeho Cho;;The Dual Effects of Interleukin-18 in Tumor Progression[J];Cellular & Molecular Immunology;2007年05期
2 ;Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B[J];Hepatobiliary & Pancreatic Diseases International;2008年05期
本文编号：2864200
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