重组人胸腺素β16的表达、纯化和胸腺素β16的应用研究
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摘要
Tβ16作为胸腺素β家族成员之一,可通过抗感染和免疫调节作用、刺激成纤维细胞的增殖、促进上皮细胞角化、刺激内皮细胞移动以及促进血管形成等作用来促进创伤修复。
本课题应用基因工程重组技术,设计构建含SUMO-Tβ16融合基因的重组质粒SUMO-Tβ16/pET3c,将该质粒转化大肠杆菌BL21(DE3)中,经克隆筛选获得了两株DNA测序完全正确的重组工程菌SUMO-Tβ16/pET3c/BL21。该工程菌连续传代100代,经各项指标检测,遗传稳定性很好。重组SUMO-Tβ16融合工程菌,经0.8mmol/L的IPTG诱导,30℃培养6h后,SDS-PAGE电泳显示为可溶性表达。通过DEAE阴离子交换层析→疏水层析→SUMO蛋白酶(Ulp1)剪切→凝胶过滤层析→获得纯度大于95%、比活性为5.3×105IU/mg的Tβ16。体内外生物学活性实验证明:Tβ16具有促进成纤维细胞增殖;促进兔角膜细胞增殖;促进鸡胚绒毛膜尿囊膜的血管增殖;Tβ16凝胶能够促进家兔皮肤烫伤和碱烧伤的愈合。
总之,本课题研究结果表明Tβ16可以通过抗感染、促进成纤维细胞增殖和促进血管的再生等作用对创伤进行修复,初步确定Tβ16有效剂量为5~10μg/克,研究结果为Tβ16药物的开发提供实验依据。
Trauma repair is a complex regulatory process which includes following phases: (1)Hemostasis.(2)Inflammation phase, which is involved with neutrophils, macrophages, lymphocytes and mastocytes.(3)Fibroblast proliferation.(4)Neovascularization, which includes endothelial cell transformation, differentiation and maturation. (5)Wound and scar hyperplasia and contraction, which is involved with myofibroblast contraction and epithelial cells proliferation.(6)Re-epithelialization, a complex dynamic process which is consisted with hom cells transformation, proliferation and maturation.
Currently, drugs for trauma repair mainly refer to growth factors, such as EGF, FGE, KGF and etc. According to their different bioactivities, the deficiencies are as follows: (1) EGF mainly participates in late re-epithelialization, while FGF mainly participates in early growth of granulation, thus bFGF is usually recommended in patient with large area of wound injury where granulation tissues deposition are needed, and KGF is usually recommended in patient with light skin injury where re-epithelialization are needed.(2) Growth factor drugs can promote cell proliferation while without any effect of anti-inflamation. (3) Most growth factors use tyrosine protein kinase system for signal transduction,which is related with cancer formation, for which FDA didn’t approve any FGF-drug for clinic applications until now.
Tβ16, consisted with 45 amino acids, having a molecular weight of 5 KDa is firstly found in human neuroblastoma cells, which wildly exists in nerve, parotid, and testis tissues and superficial cells in healthy person.
As a member of thymosin family, the functions of Tβ16 are different from other commercial available or developing drugs, which characteristics are as follows:
(1) Function combination of both anti-infection and immune regulation.
(2)Specificly stimulating proliferation of fibroblasts and improving keratinization of epithelial cells.
(3)Specificly stimulating transformation of endothelial cells and formation of blood vessel.
(4)Low molecular weight including 45 amino acids which make it easy to spread in tissues (opposite to other growth factors having high molecular weight).
(5) Simple constructions with definite and specific functions and light adverse reactions which make it can be applied locally and repeatedly.
(6) Having multiple administration routes (such as inhale and percutaneous absorption) with long effect and limited times of application as being a polypeptide drug.
(7) Participating in the construction of cytoskeleton system and the maintaince of cell construction stability.
(8) Participating in the hair follicle development process by improving hair follicle stem cells migration, differentiation and extracellular matrix reconstruction. For all above reasons, the research of Tβ16 on trauma repair is considered with important clinical significance which may be applied wildly in the future. Current research on Tβ4 synthesized by chemical method in abroad has come into phase II clinical trial. But the high costs and environment pollution during preparation limits its application extraordinarily. Tβ16 has similarly functions with Tβ4, while its research history and relative reports especially on genetic engineering are still short and rare. In this paper, the author describes the expression process of fusion protein SUMO-Tβ16 in prokaryotic expression system and the isolation and purification process technique for expressed protein through ion exchange chromatography (atmospheric pressure). It is easy to operate with low cost and fitting for industrialized manufacture.
As a small peptide, Tβ16 has only 45 animo acids that its expression product of routine expression vectors in Ecoli could be degraded easily, while its detection standards are limited, and the yield and recovery rate can not reach the qualification for industrialization. In this study, the author used fusion protein expression system for the expression of Tβ16. Moreover, with SUNO fusion tag the expression of Tβ16 has been optimized obviously, which advantages are as follows: (1) Anti proteolysis.(2) Enhancement on protein expression and maintenance of protein stability.(3) Improvement on correct protein folding and solubility.
In this study, fusion protein expression vector SUMP- Tβ16/pET3c was constructed successfully and transferred into E.coli. BL21 (DE3) for screening and sequencing. Recombinant engineering stain SUMO- Tβ16/pET3c/BL21 with correct DNA sequence of Tβ16 were passaged for 100 passages in which no significant morphological characteristic changes were observed. Target gene of the engineering strain was analysised by PCR, restriction endonuclease digestion reactions and electrophoresis, which was proved to be specific and stable; The expression level of the engineering strain was evaluated through whole stain SDS-PAGE electrophoresis, which results showed there wasn’t any significant differences among the recombinant strain of every passage; The sequence among different passage was analysised, and the result showed there existed sequence consistency among strains from the original passage to the 100th; Antigenicity of His-SUMO- Tβ16 of the expressed product among each passage was evaluated, in which Western blot tests all demonstrated positive reactions. In other relative tests, the genetic stability of the recombinant strain showed extremely well.
The study has established a fermentation and purification process technology for industrialized manufacture ofβ16. After induction by 0.8mol/L IPTG at 30℃for 6 hours, SDS-PAGE electrophoresis of the expressed product of recombinant engineering strain SUMO-Tβ16 showed soluble expression. The purification process of the protein was: DEAE anion-exchange chromatography→hydrophobic interaction chromatography→SUMO Protease digestion→Gel filtration chromatography. The purification of purified product was more than 95%, and the activity was 5.3×105 IU/mg.
The purification and digestion process in this study is easy and effective. Since SUMO tag can lead soluble expression, the expressed protein needs no denaturation and renaturation. And because SUMO tag can be recognized by SUMO enzyme and digested exactly without any extended N-ends left, many problems caused by other chemical or enzymologic digestions methods, such as low yield, formation of protein precipitation, complex digestion conditions, high cost of protease, formation of undesirable N-ends and ect by Factor Xa and thrombase are avoided. Else, protein expressed in the form of inclusion body led by GST expression tag which needs denaturation and renaturation are also avoided.
Protease SUMO (Ulp 1) used in this study was prepared by our research department. The preparation process was as follows: Analysis prepared His-Ulpl/pET3c/BL21(DE3) engineering strain to proof the correction of target DNA sequences; Induce the expression strain in high density fermentation by 1.0mmol/L IPTG at 30℃for 6 hours; Analysis the expression scale using SDS-PAGE electrophoresis, which result was 24.3%; Purify expressed Ulpl through CM anode exchange one step purification process which protein purification was more than 98% in SDS-PAGE electrophoresis analysis, which equivalently 355mg of pure Ulpl was obtained per liter fermentation broth;Evaluate the immune response between expressed Ulpl and 6xHis antibody through Western blot analysis, which results showed obvious antigenicity against 6xHis antibody. The results of in vitro or in vivo biological activity tests indicated:Τβ16 could promote cell proliferation of fibroblasts and rabbit corner cells, migration of human umbilical vein endothelial cells and vascular proliferation of chorioallantoic membrane in vitro, and the healing of rabbit skin and alkali burn in vivo effectively.
All in all, this study has primarily analysised the bioactivity ofΤβ16 in vitro and in vivo. The results indicate thatΤβ16 participate trauma repair both by anti-infection and promotion functions on fibroblast and vascular proliferation and etc. The preliminary effective dosage ofΤβ16 is 5~10μg/gram, which provides a good basis for further research.
本课题应用基因工程重组技术,设计构建含SUMO-Tβ16融合基因的重组质粒SUMO-Tβ16/pET3c,将该质粒转化大肠杆菌BL21(DE3)中,经克隆筛选获得了两株DNA测序完全正确的重组工程菌SUMO-Tβ16/pET3c/BL21。该工程菌连续传代100代,经各项指标检测,遗传稳定性很好。重组SUMO-Tβ16融合工程菌,经0.8mmol/L的IPTG诱导,30℃培养6h后,SDS-PAGE电泳显示为可溶性表达。通过DEAE阴离子交换层析→疏水层析→SUMO蛋白酶(Ulp1)剪切→凝胶过滤层析→获得纯度大于95%、比活性为5.3×105IU/mg的Tβ16。体内外生物学活性实验证明:Tβ16具有促进成纤维细胞增殖;促进兔角膜细胞增殖;促进鸡胚绒毛膜尿囊膜的血管增殖;Tβ16凝胶能够促进家兔皮肤烫伤和碱烧伤的愈合。
总之,本课题研究结果表明Tβ16可以通过抗感染、促进成纤维细胞增殖和促进血管的再生等作用对创伤进行修复,初步确定Tβ16有效剂量为5~10μg/克,研究结果为Tβ16药物的开发提供实验依据。
Trauma repair is a complex regulatory process which includes following phases: (1)Hemostasis.(2)Inflammation phase, which is involved with neutrophils, macrophages, lymphocytes and mastocytes.(3)Fibroblast proliferation.(4)Neovascularization, which includes endothelial cell transformation, differentiation and maturation. (5)Wound and scar hyperplasia and contraction, which is involved with myofibroblast contraction and epithelial cells proliferation.(6)Re-epithelialization, a complex dynamic process which is consisted with hom cells transformation, proliferation and maturation.
Currently, drugs for trauma repair mainly refer to growth factors, such as EGF, FGE, KGF and etc. According to their different bioactivities, the deficiencies are as follows: (1) EGF mainly participates in late re-epithelialization, while FGF mainly participates in early growth of granulation, thus bFGF is usually recommended in patient with large area of wound injury where granulation tissues deposition are needed, and KGF is usually recommended in patient with light skin injury where re-epithelialization are needed.(2) Growth factor drugs can promote cell proliferation while without any effect of anti-inflamation. (3) Most growth factors use tyrosine protein kinase system for signal transduction,which is related with cancer formation, for which FDA didn’t approve any FGF-drug for clinic applications until now.
Tβ16, consisted with 45 amino acids, having a molecular weight of 5 KDa is firstly found in human neuroblastoma cells, which wildly exists in nerve, parotid, and testis tissues and superficial cells in healthy person.
As a member of thymosin family, the functions of Tβ16 are different from other commercial available or developing drugs, which characteristics are as follows:
(1) Function combination of both anti-infection and immune regulation.
(2)Specificly stimulating proliferation of fibroblasts and improving keratinization of epithelial cells.
(3)Specificly stimulating transformation of endothelial cells and formation of blood vessel.
(4)Low molecular weight including 45 amino acids which make it easy to spread in tissues (opposite to other growth factors having high molecular weight).
(5) Simple constructions with definite and specific functions and light adverse reactions which make it can be applied locally and repeatedly.
(6) Having multiple administration routes (such as inhale and percutaneous absorption) with long effect and limited times of application as being a polypeptide drug.
(7) Participating in the construction of cytoskeleton system and the maintaince of cell construction stability.
(8) Participating in the hair follicle development process by improving hair follicle stem cells migration, differentiation and extracellular matrix reconstruction. For all above reasons, the research of Tβ16 on trauma repair is considered with important clinical significance which may be applied wildly in the future. Current research on Tβ4 synthesized by chemical method in abroad has come into phase II clinical trial. But the high costs and environment pollution during preparation limits its application extraordinarily. Tβ16 has similarly functions with Tβ4, while its research history and relative reports especially on genetic engineering are still short and rare. In this paper, the author describes the expression process of fusion protein SUMO-Tβ16 in prokaryotic expression system and the isolation and purification process technique for expressed protein through ion exchange chromatography (atmospheric pressure). It is easy to operate with low cost and fitting for industrialized manufacture.
As a small peptide, Tβ16 has only 45 animo acids that its expression product of routine expression vectors in Ecoli could be degraded easily, while its detection standards are limited, and the yield and recovery rate can not reach the qualification for industrialization. In this study, the author used fusion protein expression system for the expression of Tβ16. Moreover, with SUNO fusion tag the expression of Tβ16 has been optimized obviously, which advantages are as follows: (1) Anti proteolysis.(2) Enhancement on protein expression and maintenance of protein stability.(3) Improvement on correct protein folding and solubility.
In this study, fusion protein expression vector SUMP- Tβ16/pET3c was constructed successfully and transferred into E.coli. BL21 (DE3) for screening and sequencing. Recombinant engineering stain SUMO- Tβ16/pET3c/BL21 with correct DNA sequence of Tβ16 were passaged for 100 passages in which no significant morphological characteristic changes were observed. Target gene of the engineering strain was analysised by PCR, restriction endonuclease digestion reactions and electrophoresis, which was proved to be specific and stable; The expression level of the engineering strain was evaluated through whole stain SDS-PAGE electrophoresis, which results showed there wasn’t any significant differences among the recombinant strain of every passage; The sequence among different passage was analysised, and the result showed there existed sequence consistency among strains from the original passage to the 100th; Antigenicity of His-SUMO- Tβ16 of the expressed product among each passage was evaluated, in which Western blot tests all demonstrated positive reactions. In other relative tests, the genetic stability of the recombinant strain showed extremely well.
The study has established a fermentation and purification process technology for industrialized manufacture ofβ16. After induction by 0.8mol/L IPTG at 30℃for 6 hours, SDS-PAGE electrophoresis of the expressed product of recombinant engineering strain SUMO-Tβ16 showed soluble expression. The purification process of the protein was: DEAE anion-exchange chromatography→hydrophobic interaction chromatography→SUMO Protease digestion→Gel filtration chromatography. The purification of purified product was more than 95%, and the activity was 5.3×105 IU/mg.
The purification and digestion process in this study is easy and effective. Since SUMO tag can lead soluble expression, the expressed protein needs no denaturation and renaturation. And because SUMO tag can be recognized by SUMO enzyme and digested exactly without any extended N-ends left, many problems caused by other chemical or enzymologic digestions methods, such as low yield, formation of protein precipitation, complex digestion conditions, high cost of protease, formation of undesirable N-ends and ect by Factor Xa and thrombase are avoided. Else, protein expressed in the form of inclusion body led by GST expression tag which needs denaturation and renaturation are also avoided.
Protease SUMO (Ulp 1) used in this study was prepared by our research department. The preparation process was as follows: Analysis prepared His-Ulpl/pET3c/BL21(DE3) engineering strain to proof the correction of target DNA sequences; Induce the expression strain in high density fermentation by 1.0mmol/L IPTG at 30℃for 6 hours; Analysis the expression scale using SDS-PAGE electrophoresis, which result was 24.3%; Purify expressed Ulpl through CM anode exchange one step purification process which protein purification was more than 98% in SDS-PAGE electrophoresis analysis, which equivalently 355mg of pure Ulpl was obtained per liter fermentation broth;Evaluate the immune response between expressed Ulpl and 6xHis antibody through Western blot analysis, which results showed obvious antigenicity against 6xHis antibody. The results of in vitro or in vivo biological activity tests indicated:Τβ16 could promote cell proliferation of fibroblasts and rabbit corner cells, migration of human umbilical vein endothelial cells and vascular proliferation of chorioallantoic membrane in vitro, and the healing of rabbit skin and alkali burn in vivo effectively.
All in all, this study has primarily analysised the bioactivity ofΤβ16 in vitro and in vivo. The results indicate thatΤβ16 participate trauma repair both by anti-infection and promotion functions on fibroblast and vascular proliferation and etc. The preliminary effective dosage ofΤβ16 is 5~10μg/gram, which provides a good basis for further research.
引文
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