生物抗菌肽的研究及葡萄糖耐量因子对鼠肝胰岛素受体基因表达的影响
摘要
抗菌肽(antimicrobial peptides)是多种动物对微生物等因素侵染产生的免疫应答反应的产物，具有分子量低、热稳定、强碱性和广谱抗菌等特点。其独特的抗菌机理能在细胞质膜上形成离子通道，引起胞内物质泄露，从而杀灭细菌。当今抗生素药物是人类对付细菌等病原体引起的各类疾病的主要手段，但病原微生物的抗药性已成为不容忽视的问题。抗菌肽由于其抑制或杀灭病原微生物的特殊机理消除了病原微生物易产生抗药性这一问题，从而最有希望成为补充和替代抗生素的新型药物。由于从动物组织难于获取大量天然抗菌肽，因此利用基因工程进行抗菌肽药物开发成为人们关注的焦点。
本实验主要研究的是Magainin-Protegrin杂合抗菌肽基因工程菌的构建、表达、纯化及活性测定。Magainins是爪蟾的皮肤在一定的环境压力下分泌出的抗感染和促进伤口愈合的成分，低浓度便可抑制许多细菌和真菌生长；Protegrin是1993年从猪的中性白细胞中分离得到，研究发现它具有抗爱滋病毒的功能。
该实验通过取Magainin和Protegrin主要生物活性的氨基酸序列并人工合成其编码基因，在合成基因的两侧添加适当的酶切位点。利用现有蛋白结构预测软件进行分析避免空间位阻，然后在两个小的肽链之间加上起连接作用的3个氨基酸。将该序列连接到pGEX-4T-1表达载体上构成重组质粒pGEX-4T-1-MP，将该质粒转化到E.coli BL21 (DE3) 37℃在LB培养基中培养，至OD600=0.6-0.8，加入浓度为0.75mM的IPTG诱导表达。离心收集菌体，超声破碎处理后，4℃。14，000rpm离心收集上清，获得以可溶性形式存在的表达产物。Tris-Gly-SDS-PAGE分析表明，在31KD处出现明显的表达条带。表达的蛋白经亲和柱层析，纯度达90％，最后进行了抑菌检测。
另外本文还研究了实验小鼠注射复方葛根胶囊(主要成分是葡萄糖耐量因子GTF)后对肝脏内胰岛素受体基因表达的影响。以Cr~(3+)为主组成的GTF能把胰岛素结合到膜受体上并促进胰岛素活性增强，能促进糖代谢和脂代谢。该实验通过提取小鼠肝脏的RNA，反转录后PCR半定量分析胰岛素受体基因的表达情况，结果表明用药后有明显效果。
Antimicrobial peptides are the immune response products of animals infected by the microbes. The antimicrobial pptides have many characters such as low molecular weight, thermostability, strong alkalinity and wide antibiogram. The ion channels on the cell membrane can be formed because of their distinctive mechanisms of killing microbes, and that cause the cell content to leak out and finally lead the bacteria to die. Antibiotic is the main way to deal with the diseases caused by the pathogens nowadays, but the resistances of microbes to the antibiotics are becoming the more and more serious problem that cannot be ignored. The resistance of microbes to antimicrobial peptides cann't be formed because of their special mechanisms of killing the microbes. So the antimicrobial peptides are the most prospective substitutes for the antibiotics. Because it is difficult to obtain antimicrobial peptides in large scales from animal tissues, the production of antimicrobial peptides by genetic engineering becomes the focus of people's interests.
The subject in this study is the recombination , expression, purification and biological activity on antibacterial fusion peptide of, Magainin-Protegrin . Magainin is the skin secretion product of Batrachia under the eriviroment pressure, which can be used to accelerate the fusion rate of wound and resist the infection. It can inhibit the growth of bacteria and fungus with the lowest concentration; Protegrin is isolated from porcine leukocyte hi 1993 and is useful as antiviral agents.
Firstly, the amino acid sequence of major bioactivity hi Magainin and Pro-tegrinin have been founded from the Genebank and then the related gene has been artifically synthesized. The enzyme sites are added to the two sides of the synthesized gene. The two peptides are connected with the three amino acid after the analysis of the software on the predication of the protein structure to avoid the interference hi the space. The PCR product was cut by two restriction enzymes, purified and cloned into the vector pGEX-4T-1. The engineering strains were incubated at 37 until OD600 ,0.6-0.8, then IPTG was added to reach the concentration of 0.75mmol/L to induce the fusion expression of the functional gene at 37 for 7 h. Tris-Gly-SDS-PAGE assay suggested the bacteria harboring the recombinant plasmid pGEX-4T-l- MP produce a fusion protein of 31KD. The purity of expressed protein after treatment by GST affinity chromatography reached 90% and at last the assay of inhibiting bacteria has been conducted.
We also studied the effect on gene expression of the mouse liver cell which was dealt with the medicine of capsule of Fufang Gegen (the major component is the Glucose Tolerance Factor GTF). The main composition -chromium(3+) plays the important part in the increasement of insulin activity ,the enhencement of metabolism rate of sugar and fat , and the combination of insulin and the receptor of membrane. RNA has been extracted from the liver cell and the expression has been analyzed by the half-quantum after RT-PCR. The result displays the good effect of the medicine.
本实验主要研究的是Magainin-Protegrin杂合抗菌肽基因工程菌的构建、表达、纯化及活性测定。Magainins是爪蟾的皮肤在一定的环境压力下分泌出的抗感染和促进伤口愈合的成分，低浓度便可抑制许多细菌和真菌生长；Protegrin是1993年从猪的中性白细胞中分离得到，研究发现它具有抗爱滋病毒的功能。
该实验通过取Magainin和Protegrin主要生物活性的氨基酸序列并人工合成其编码基因，在合成基因的两侧添加适当的酶切位点。利用现有蛋白结构预测软件进行分析避免空间位阻，然后在两个小的肽链之间加上起连接作用的3个氨基酸。将该序列连接到pGEX-4T-1表达载体上构成重组质粒pGEX-4T-1-MP，将该质粒转化到E.coli BL21 (DE3) 37℃在LB培养基中培养，至OD600=0.6-0.8，加入浓度为0.75mM的IPTG诱导表达。离心收集菌体，超声破碎处理后，4℃。14，000rpm离心收集上清，获得以可溶性形式存在的表达产物。Tris-Gly-SDS-PAGE分析表明，在31KD处出现明显的表达条带。表达的蛋白经亲和柱层析，纯度达90％，最后进行了抑菌检测。
另外本文还研究了实验小鼠注射复方葛根胶囊(主要成分是葡萄糖耐量因子GTF)后对肝脏内胰岛素受体基因表达的影响。以Cr~(3+)为主组成的GTF能把胰岛素结合到膜受体上并促进胰岛素活性增强，能促进糖代谢和脂代谢。该实验通过提取小鼠肝脏的RNA，反转录后PCR半定量分析胰岛素受体基因的表达情况，结果表明用药后有明显效果。
Antimicrobial peptides are the immune response products of animals infected by the microbes. The antimicrobial pptides have many characters such as low molecular weight, thermostability, strong alkalinity and wide antibiogram. The ion channels on the cell membrane can be formed because of their distinctive mechanisms of killing microbes, and that cause the cell content to leak out and finally lead the bacteria to die. Antibiotic is the main way to deal with the diseases caused by the pathogens nowadays, but the resistances of microbes to the antibiotics are becoming the more and more serious problem that cannot be ignored. The resistance of microbes to antimicrobial peptides cann't be formed because of their special mechanisms of killing the microbes. So the antimicrobial peptides are the most prospective substitutes for the antibiotics. Because it is difficult to obtain antimicrobial peptides in large scales from animal tissues, the production of antimicrobial peptides by genetic engineering becomes the focus of people's interests.
The subject in this study is the recombination , expression, purification and biological activity on antibacterial fusion peptide of, Magainin-Protegrin . Magainin is the skin secretion product of Batrachia under the eriviroment pressure, which can be used to accelerate the fusion rate of wound and resist the infection. It can inhibit the growth of bacteria and fungus with the lowest concentration; Protegrin is isolated from porcine leukocyte hi 1993 and is useful as antiviral agents.
Firstly, the amino acid sequence of major bioactivity hi Magainin and Pro-tegrinin have been founded from the Genebank and then the related gene has been artifically synthesized. The enzyme sites are added to the two sides of the synthesized gene. The two peptides are connected with the three amino acid after the analysis of the software on the predication of the protein structure to avoid the interference hi the space. The PCR product was cut by two restriction enzymes, purified and cloned into the vector pGEX-4T-1. The engineering strains were incubated at 37 until OD600 ,0.6-0.8, then IPTG was added to reach the concentration of 0.75mmol/L to induce the fusion expression of the functional gene at 37 for 7 h. Tris-Gly-SDS-PAGE assay suggested the bacteria harboring the recombinant plasmid pGEX-4T-l- MP produce a fusion protein of 31KD. The purity of expressed protein after treatment by GST affinity chromatography reached 90% and at last the assay of inhibiting bacteria has been conducted.
We also studied the effect on gene expression of the mouse liver cell which was dealt with the medicine of capsule of Fufang Gegen (the major component is the Glucose Tolerance Factor GTF). The main composition -chromium(3+) plays the important part in the increasement of insulin activity ,the enhencement of metabolism rate of sugar and fat , and the combination of insulin and the receptor of membrane. RNA has been extracted from the liver cell and the expression has been analyzed by the half-quantum after RT-PCR. The result displays the good effect of the medicine.
引文
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2. Numao.N, et al. Biochem-Pharmacol,1999, Jan 15:57(2):121-134.
3．赵东红，戴祝英，周开亚．昆虫抗菌肽的功能，作用机理与分子生物学研究最新进展．生物工程进展，1999，19(5)：14-17。
4．许玉澄，张双全，等．家蚕抗菌肽的抗癌作用．动物学研究，1998，19(4)：263-268．
5. Boman H G et al. Antibacterial Peptides: Key Components Needed in Immunity. Cell, 1991, 65:205-207.
6．石强，刘飞鹏．抗菌肽克隆基因的表达和转基因研究现状．生物工程进展，2000，20(1)：37-40．
7. Oren. Z, Shai Y, A class of highly potent antibacterial peptides derived from pardaxin, a pore-forming peptide isolated from moses sole fish pardachirus marmoratus. Eur J Biochem, 1996,237(1):303-310.
8. Holak, T.A, Linderberg.G, et al; Biochemistry, 1998,27:7620-7629.
9．崔晓江，刘枝俏，等．杀菌肽的研究进展及应用前景．生物化学与生物物理进展，1995，22(1)：5-8．
10. Jaynes J M. Drug news and perspectives, 1990,3(2):69-72.
11. Zasloff M, et al. Magainins, a class of antimicrobial peptides from Xenopus skin isolation, characterization of two active forms, and partial cDNA sequence of a precursor [J]. Proc Natl Acad Sci USA, 1987; 84: 5449-5455.
12. Yamasaki M. Antitumor and antimicrobial glycoproteins from sea hares. Comp Biochem Physic,1993,105,(2):141-146.
13．徐强，华跃进，等．蛙类皮肤分泌物中的抗菌肽和抗癌肽．动物学杂志，2002，37(2)：73-75．
14．徐进署，张双全．动物抗菌肽．生命的化学，2001，21(2)：144-145．
15. B.Bechinger, M.Zasloff, et al. Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy, Protein Sci. 2(1993)2077-2084.
16. B Agerberth, JY Lee, et al. Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides. European Journal of Biochemistry,1991,202:849-854.
17．郭玉梅等．某些抗菌肽的的研究进展．生命科学，1997，9(1)：37-39．
18．陈留存，王金星．昆虫抗菌肽研究现状．生物工程进展，1999，19(5)：55-59．
19．叶显，白俊杰．抗菌肽的研究及其在水产上的应用前景．珠江水产，2000，2，8-12．
20．饶贤才，胡福泉等．肽抗生素，控制感染的新希望．生命的化学，2001，21：357-359．
21. Intra Biotics Pharmaceuticals, Inc. Development of Protegrins. Biopolymers peptide
science, 2000, vol.55,88-98.
22. James P, Chengwe Wu, et al. Membrane selectivity of cystine-stabilized cylic protegrins. Euro. J. Biochem, 267,3289-3300.
23．松崎腾已．作用于生物膜的抗菌肽分子学作用机制．国外医学药学分册，1998，25(2)：107-109．
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26. Juvvadi P, et al. Journal of peptide science, 1996,2(4),:223-32.
27. Park CB, et al. Source Biochemical&Biophysical research communication, 1998, 224(1): 253-257.
28. Lee D G, et al. Biochemistry&molecular Biology International, 1998, 43(3):489-98.
29. Ramos-onsins-s, Aguade-M. Genetics, 1998, Sep, 150(1):15—171.
30. Kim-Hs, Park-Cb. Biochem-Biophys-Res-Commun, 1998, may, 19. 246(2):388-92.
31．谢毅，闵水洁，贾士荣．生物工程学报，1990，6(4)，311-315．
32．李秀兰，戴祝英，张双全，等．家蚕抗菌肽CMIV基因结构改造及表达产物的研究．中国生物化学与分子生物学报，1999，15(3)：387-391．
33．吴映雅，刘飞鹏，周天鸿，等．抗菌肽与碱性成纤维细胞生长因子基因的融合及其在酵母中的表达．生物工程进展，1998，18(5)：11-14．
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37．黄大年，朱冰，田文忠，等．抗菌肽B基因导入水稻及转基因植株的鉴定．中国科学，1997，27(1)：55-62．
38．李乃坚，袁四清，黄自然．农业生物技术学报，1998，6(2)：178-184．
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41．郭玉梅，戴祝英．昆虫抗菌肽的研究进展．生物工程进展，1999，6(3)：24-28．
42．黄自然．科学通报，1998，14：1107-1109．
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2. Numao.N, et al. Biochem-Pharmacol,1999, Jan 15:57(2):121-134.
3．赵东红，戴祝英，周开亚．昆虫抗菌肽的功能，作用机理与分子生物学研究最新进展．生物工程进展，1999，19(5)：14-17。
4．许玉澄，张双全，等．家蚕抗菌肽的抗癌作用．动物学研究，1998，19(4)：263-268．
5. Boman H G et al. Antibacterial Peptides: Key Components Needed in Immunity. Cell, 1991, 65:205-207.
6．石强，刘飞鹏．抗菌肽克隆基因的表达和转基因研究现状．生物工程进展，2000，20(1)：37-40．
7. Oren. Z, Shai Y, A class of highly potent antibacterial peptides derived from pardaxin, a pore-forming peptide isolated from moses sole fish pardachirus marmoratus. Eur J Biochem, 1996,237(1):303-310.
8. Holak, T.A, Linderberg.G, et al; Biochemistry, 1998,27:7620-7629.
9．崔晓江，刘枝俏，等．杀菌肽的研究进展及应用前景．生物化学与生物物理进展，1995，22(1)：5-8．
10. Jaynes J M. Drug news and perspectives, 1990,3(2):69-72.
11. Zasloff M, et al. Magainins, a class of antimicrobial peptides from Xenopus skin isolation, characterization of two active forms, and partial cDNA sequence of a precursor [J]. Proc Natl Acad Sci USA, 1987; 84: 5449-5455.
12. Yamasaki M. Antitumor and antimicrobial glycoproteins from sea hares. Comp Biochem Physic,1993,105,(2):141-146.
13．徐强，华跃进，等．蛙类皮肤分泌物中的抗菌肽和抗癌肽．动物学杂志，2002，37(2)：73-75．
14．徐进署，张双全．动物抗菌肽．生命的化学，2001，21(2)：144-145．
15. B.Bechinger, M.Zasloff, et al. Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy, Protein Sci. 2(1993)2077-2084.
16. B Agerberth, JY Lee, et al. Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides. European Journal of Biochemistry,1991,202:849-854.
17．郭玉梅等．某些抗菌肽的的研究进展．生命科学，1997，9(1)：37-39．
18．陈留存，王金星．昆虫抗菌肽研究现状．生物工程进展，1999，19(5)：55-59．
19．叶显，白俊杰．抗菌肽的研究及其在水产上的应用前景．珠江水产，2000，2，8-12．
20．饶贤才，胡福泉等．肽抗生素，控制感染的新希望．生命的化学，2001，21：357-359．
21. Intra Biotics Pharmaceuticals, Inc. Development of Protegrins. Biopolymers peptide
science, 2000, vol.55,88-98.
22. James P, Chengwe Wu, et al. Membrane selectivity of cystine-stabilized cylic protegrins. Euro. J. Biochem, 267,3289-3300.
23．松崎腾已．作用于生物膜的抗菌肽分子学作用机制．国外医学药学分册，1998，25(2)：107-109．
24. Christensen B, et al. Proceedings of national Academy sciences of the united states of America, 1998, vol.85, 5072-5076.
25. Lockey T.D, et al. European Journal of Biochemistry, 1996, 236(1):263-271.
26. Juvvadi P, et al. Journal of peptide science, 1996,2(4),:223-32.
27. Park CB, et al. Source Biochemical&Biophysical research communication, 1998, 224(1): 253-257.
28. Lee D G, et al. Biochemistry&molecular Biology International, 1998, 43(3):489-98.
29. Ramos-onsins-s, Aguade-M. Genetics, 1998, Sep, 150(1):15—171.
30. Kim-Hs, Park-Cb. Biochem-Biophys-Res-Commun, 1998, may, 19. 246(2):388-92.
31．谢毅，闵水洁，贾士荣．生物工程学报，1990，6(4)，311-315．
32．李秀兰，戴祝英，张双全，等．家蚕抗菌肽CMIV基因结构改造及表达产物的研究．中国生物化学与分子生物学报，1999，15(3)：387-391．
33．吴映雅，刘飞鹏，周天鸿，等．抗菌肽与碱性成纤维细胞生长因子基因的融合及其在酵母中的表达．生物工程进展，1998，18(5)：11-14．
34. Jaynes J M, et al. Plant Science, 1993, 89, 43-53.
35. Gatz.C, Lenk.I, et al. Trends in plant science 1998, 3: 252-258.
36．贾士荣，屈贤明．马铃薯抗菌肽基因工程．北京：农业科学出版社，1996．
37．黄大年，朱冰，田文忠，等．抗菌肽B基因导入水稻及转基因植株的鉴定．中国科学，1997，27(1)：55-62．
38．李乃坚，袁四清，黄自然．农业生物技术学报，1998，6(2)：178-184．
39. Reed-WA, Elzer-PH, Jayres-JM. Transgenic-Res, 1997, 6(5):337-347.
40. David-Winder, Walter H, et al. Biochem-Biophys-Res-Commu, 1998,242:608-612.
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42．黄自然．科学通报，1998，14：1107-1109．
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