重組人白介素24在大腸桿菌中的高效表達及其抗腫瘤應用
發(fā)布時間：2020-10-31 16:29
P. B. Fisher等人在細胞表面受體Mda-7發(fā)現(xiàn)的基礎上，鑒定了黑素瘤分化相關蛋白-7（MDA-7），隨后MDA-7被重命名為IL-24。IL24屬于IL-10細胞因子家族成員，該家族包括IL-10、IL-19、IL-20、IL-22、IL-24和IL-26。IL-24具有抑制多種人類癌細胞生長的特性，同時對正常細胞無誘導危害。IL-24具有潛在的治療應用價值并在腫瘤細胞學領域起著重要作用。 重組人源IL-24（rhIL-24）最初是使用Escherichia coli進行生產(chǎn)并利用傳統(tǒng)方法進行純化鑒定，其發(fā)酵策略是基于LB培養(yǎng)基的分批培養(yǎng)，然后利用高壓均質機來裂解細胞。在E.coli中表達真核生物蛋白會導致不溶性的包涵體（IBs）的形成。對活性治療性蛋白生產(chǎn)來說，包涵體的溶解和重折疊是一個關鍵挑戰(zhàn)。傳統(tǒng)的包涵體溶解和重折疊方法純化應用于小體積的樣品，使用陰離子和陽離子交換色譜法從樣品中取出雜質，從而得到純化的產(chǎn)品。這種傳統(tǒng)的rhIL-24制備方法被認為是一個耗時、費力的過程。本文設計了一種基于傳統(tǒng)方法的高效、低成本的rhIL-24制備策略。培養(yǎng)基的成本和組成對于商業(yè)規(guī)模的E.coli重組蛋白生產(chǎn)至關重要，采用向培養(yǎng)基中添加酵母提取物、葡萄糖等成分提高rhIL-24的產(chǎn)量；同時，用乳糖替代IPTG的使用，也降低了成本和毒性。此外，我們用一種2步變性-重折疊法（2DR）替代傳統(tǒng)的重折疊法，從而提高了rhIL-24的產(chǎn)量。新的制備策略使得高質量、高純度的rhIL-24獲得過程更加簡單，同時，LC-MS/MS技術的運用為rhIL-24提供了精確的定性、定量信息。 一步純化的rhIL-24被作為癌癥和多藥耐藥腫瘤細胞的有效的治療劑進行相關分析。rhIL-24在肝癌細胞系HepG2顯示了生物活性，但是對L02細胞沒有影響。膜上的P-糖蛋白（P-gp）是多藥耐藥的主要因素。我們研究了rhIL-24對抗阿霉素（ADM）人源乳腺癌細胞系MCF-7/ADM的影響，利用MTT法對rhIL24和ADM的細胞毒性進行了檢測，P-gp的表達用顯微共聚焦法和Western blot法分析評估，ADM在MCF-7/ADM細胞系中的IC50在加入rhIL-24后呈現(xiàn)劑量依賴性降低。在低劑量的rhIL-24（(4μPol·L-1)）的MCF-7/ADM細胞系中，ADM積累增加，而P-gp的表達降低。體外生物活性表明，rhIL-24通過激活Stat3轉錄因子繞過MCF-7/ADM細胞的多藥耐藥，rhIL-24可以作為P-糖蛋白的抑制劑，用以逆轉乳腺癌細胞的阿霉素抗性。 本研究的最終目標是將rhIL-24作為可控釋放藥物�；诟杉毎幕蛑委煼桨�，受限于IL-24到皮下腫瘤的轉運。腺病毒介導的mda-7/IL-24(Ad.mda-7)法會導致過度表達引起細胞生長停滯和凋亡。在病毒載體相比，非病毒載體系統(tǒng)具有毒性低，生物相容性，可控性。非病毒載體與藥物的共軛聚合物可以顯著增加藥物的水溶性，改性的組織分布和血漿循環(huán)半衰期。蛋白質-PEG復合物已能通過增加藥物的穩(wěn)定性顯著提高療效。本研究開發(fā)的策略，產(chǎn)生一個均勻的、單體嵌合的rhIL-24和PEG魚精蛋白復合物。PEG魚精蛋白-IL24納米顆粒能夠確認對MCF-7／ADM細胞活性。PEG修飾的魚精蛋白–rhIL-24納米顆粒具有無可比擬的療效。目前的研究對一種rhIL-24控釋制劑的臨床實現(xiàn)鋪平了道路。
【學位單位】：江南大學
【學位級別】：博士
【學位年份】：2014
【中圖分類】：R378.21
【文章目錄】：
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
【參考文獻】
本文編號：2864200
【學位單位】：江南大學
【學位級別】：博士
【學位年份】：2014
【中圖分類】：R378.21
【文章目錄】：
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
【參考文獻】
相關期刊論文 前2條
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
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