兽疫链球菌合成透明质酸相关基因的克隆与表达
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摘要
透明质酸是一种由葡萄糖醛酸和N-乙酰氨基葡萄糖以β-1-3和β-1-4糖昔键相连的二糖单位重复构建而成的,具有生物相容性的高分子聚合物。由于特殊的生理作用、独特的流变学特性和极强的持水保湿能力,透明质酸在医学研究、临床治疗、化妆品工业等领域有广泛的应用。
透明质酸广泛分布于各种动物组织中,一些兼性厌氧革兰氏阳性链球菌,如链球菌属A群(group A streptococcus,GAS),C群(group Cstreptococcus,GCS)的菌膜中也有透明质酸存在。由于动物组织的原料有限,且透明质酸的含量低,同时提取成本高,因而价格十分昂贵。而微生物发酵法,所需的原料易得,大大降低了生产成本。
本研究首先对兽疫链球菌(Streptococcus Zooepidemicus)中与透明质酸合成有关的基因做了研究,主要包括透明质酸合成酶基因和磷脂酰甘油磷酸合成酶基因以及透明质酸分解酶基因。
透明质酸合成酶基因是生物合成HA途径中的关键酶,其活性和稳定性决定了透明质酸产量和质量,它还参与HA输出到胞外。对实验室保存的兽疫链球菌的透明质酸合成酶基因的进行了克隆及测序,通过序列分析,证明其属于合成透明质酸活性较高的合成酶,同时也结果验证了实验室保存生产透明质酸的链球菌为兽疫链球菌。
分离得到兽疫链球菌的磷脂酰甘油磷酸合成酶基因序列。序列分析显示:其开放阅读框为531的碱基,序列编码的蛋白含有176个氨基酸残基,分子量为19400,等电点为8.8,其中含有63个疏水氨基酸,预示其可能膜结合特性。二级结构预测发现该蛋白含3个跨膜域。此外,该蛋白与已知的产脓链球菌(Streptococcus pyogenes)的磷脂酰甘油磷酸合成酶具有85%的氨基酸序列相似性。磷脂酰甘油磷酸合成酶基因,它与心磷脂的合成有密切关系,心磷脂是对透明质酸合成酶的生物活性及稳定性有重要影响。它的分离对提高透明质酸合成酶活性,对提高透明质酸产量及提高分子量提供理论基础。对本实验室保存的兽疫链球菌改造提供了一个方向。
透明质酸分解酶是是一种胞外酶,能够裂解透明质酸,会降低发酵生产透明质酸的产量。课题研究了实验室保存的兽疫链球菌是否含有透明质酸分解酶基因,从序列分析结果来看,实验室保存的兽疫链球菌不同于其他已经诱变工业菌种,它是含有透明质酸分解酶基因的。
Hyaluronic acid(HA)is a high molecular weight linear polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked byβ-1-3 andβ-1-4 glycoside bonds.Because of its special physiological action,extraordinary rheological property and moister-holding function,HA is extensively used for medical research,clinical therapy,and cosmetic industry.
Owing to shortage of material source,high cost,and low output in traditional HA production by extraction of animal tissues,microbial fermentation has currently become the research hot spot.HA has high affinity to combine with water,and its water solution has a property of high viscosity. For this reason,HA can be added to cosmetis to keep the moisture of skin. Besides,HA can be applied to ophthalmology,arthritis heating and other medical areas.HA exists in connective tissues of mammals and the capsule of group A and group C of streptococci.
The aim of this study was to explore the HA production by Streptococci zooepidemicus.Some genes for HA biosynthesis from Streptococcus zooepidemicus were studied,including genes encoding HA synthase and phosphatidylglycerophosphate synthase and hyaluronidase.
HA synthase is a crucial enzyme in HA synthesis,its activity and stability affected the quantity and quality of HA.We cloned the gene by PCR from streptococcuz zooepemicus deposited in our lab.By sequence analysis,it was found that its putative protein has high enzyme activity in HA biosynthesis, and also identified this streptococcus strain as streptococcuz zooepidemicus.
Besides,the gene encoding phosphatidylglycerophosphate synthase(PGPS)was isolated by PCR.Through sequence analysis,it was found that its largest open reading frame was 531bp in length and the putative protein comprised 176 amonia acid residues with predicted molecular weight of 19.4 kDa and isoelectric point of 8.8.In addition,63 hydrophobic amino acids were presented in this protein,implying the property of membrane-bound protein.Transmembrane prediction revealed three transmembrane domains residing in this protein.Moreover,the putative protein shares 85%similarity with PGPS of S.pyogenes.This gene has close relation with the synthesis of cardiolipin,which is very important for the activity and stability of HA synthase.Isolation and cloning of the gene have positive affect for improving activity and stability of HA synthase,and offer a idea for improving HA production of streptococcuz zooepidemicus preserved in our lab.
Hyaluronidase is a enzyme that can decompose HA,it can decrease fermentation yield of HA.We study a streptococcuz zooepidemicus preserved in our lab and detect hyaluronidase gene wether in the genome of the bacterium,according the sequence analysis,the bacterium is different for the others mutated for industry production,containing the hyaluronidase gene.
透明质酸广泛分布于各种动物组织中,一些兼性厌氧革兰氏阳性链球菌,如链球菌属A群(group A streptococcus,GAS),C群(group Cstreptococcus,GCS)的菌膜中也有透明质酸存在。由于动物组织的原料有限,且透明质酸的含量低,同时提取成本高,因而价格十分昂贵。而微生物发酵法,所需的原料易得,大大降低了生产成本。
本研究首先对兽疫链球菌(Streptococcus Zooepidemicus)中与透明质酸合成有关的基因做了研究,主要包括透明质酸合成酶基因和磷脂酰甘油磷酸合成酶基因以及透明质酸分解酶基因。
透明质酸合成酶基因是生物合成HA途径中的关键酶,其活性和稳定性决定了透明质酸产量和质量,它还参与HA输出到胞外。对实验室保存的兽疫链球菌的透明质酸合成酶基因的进行了克隆及测序,通过序列分析,证明其属于合成透明质酸活性较高的合成酶,同时也结果验证了实验室保存生产透明质酸的链球菌为兽疫链球菌。
分离得到兽疫链球菌的磷脂酰甘油磷酸合成酶基因序列。序列分析显示:其开放阅读框为531的碱基,序列编码的蛋白含有176个氨基酸残基,分子量为19400,等电点为8.8,其中含有63个疏水氨基酸,预示其可能膜结合特性。二级结构预测发现该蛋白含3个跨膜域。此外,该蛋白与已知的产脓链球菌(Streptococcus pyogenes)的磷脂酰甘油磷酸合成酶具有85%的氨基酸序列相似性。磷脂酰甘油磷酸合成酶基因,它与心磷脂的合成有密切关系,心磷脂是对透明质酸合成酶的生物活性及稳定性有重要影响。它的分离对提高透明质酸合成酶活性,对提高透明质酸产量及提高分子量提供理论基础。对本实验室保存的兽疫链球菌改造提供了一个方向。
透明质酸分解酶是是一种胞外酶,能够裂解透明质酸,会降低发酵生产透明质酸的产量。课题研究了实验室保存的兽疫链球菌是否含有透明质酸分解酶基因,从序列分析结果来看,实验室保存的兽疫链球菌不同于其他已经诱变工业菌种,它是含有透明质酸分解酶基因的。
Hyaluronic acid(HA)is a high molecular weight linear polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked byβ-1-3 andβ-1-4 glycoside bonds.Because of its special physiological action,extraordinary rheological property and moister-holding function,HA is extensively used for medical research,clinical therapy,and cosmetic industry.
Owing to shortage of material source,high cost,and low output in traditional HA production by extraction of animal tissues,microbial fermentation has currently become the research hot spot.HA has high affinity to combine with water,and its water solution has a property of high viscosity. For this reason,HA can be added to cosmetis to keep the moisture of skin. Besides,HA can be applied to ophthalmology,arthritis heating and other medical areas.HA exists in connective tissues of mammals and the capsule of group A and group C of streptococci.
The aim of this study was to explore the HA production by Streptococci zooepidemicus.Some genes for HA biosynthesis from Streptococcus zooepidemicus were studied,including genes encoding HA synthase and phosphatidylglycerophosphate synthase and hyaluronidase.
HA synthase is a crucial enzyme in HA synthesis,its activity and stability affected the quantity and quality of HA.We cloned the gene by PCR from streptococcuz zooepemicus deposited in our lab.By sequence analysis,it was found that its putative protein has high enzyme activity in HA biosynthesis, and also identified this streptococcus strain as streptococcuz zooepidemicus.
Besides,the gene encoding phosphatidylglycerophosphate synthase(PGPS)was isolated by PCR.Through sequence analysis,it was found that its largest open reading frame was 531bp in length and the putative protein comprised 176 amonia acid residues with predicted molecular weight of 19.4 kDa and isoelectric point of 8.8.In addition,63 hydrophobic amino acids were presented in this protein,implying the property of membrane-bound protein.Transmembrane prediction revealed three transmembrane domains residing in this protein.Moreover,the putative protein shares 85%similarity with PGPS of S.pyogenes.This gene has close relation with the synthesis of cardiolipin,which is very important for the activity and stability of HA synthase.Isolation and cloning of the gene have positive affect for improving activity and stability of HA synthase,and offer a idea for improving HA production of streptococcuz zooepidemicus preserved in our lab.
Hyaluronidase is a enzyme that can decompose HA,it can decrease fermentation yield of HA.We study a streptococcuz zooepidemicus preserved in our lab and detect hyaluronidase gene wether in the genome of the bacterium,according the sequence analysis,the bacterium is different for the others mutated for industry production,containing the hyaluronidase gene.
引文
[1 ]. Meyer K, Palmer JW. The polysaccharide of the vitreous humor [J]. Biochem, 1934, 107: 629-634
[2]. Scott JE, Heatley F. Hyaluronan forms specific stable tertiary structures in aqueous solution:a 13C NMR study[J]. Nature Rev. Cancer., 2004,4: 528-539
[3]. Balazs EA. Ultrapure hyaluronic acid and the use thereof[P]. U.S. Patent, 4141973. 1979
[4]. Fraser JR, Laurent TC. Turnover and metabolism of hyaluronan[J]. Ciba Found. Symp., 1989,143:41-53
[5]. Scott JE, Cummings C, Brass A, Chen Y. Secondary and tertiary structures of hyaluronan in aqueous solution,investigated by rotary shadowing-electron microscopy and computer simulation[J]. Biochem., 1991, 274: 699-705
[6]. Prehm P. Hyaluronate is synthesized at plasma mernbranes[J]. Biochem., 1984, 220: 597-600
[7]. Lesley J, Howes N, Perschl A, Hyman R. Hyaluronan binding function of CD44 is transiently activated on T cells during an in vivo immune response[J]. Biochem., 1983, 211: 181 -189
[8]. Balazs EA, Denlinger JL. Viscosupple mentation: A new concept in the treatment of osteoarthritis[J]. J Rheumatol, 1993, 39: 3-9
[9]. Madsen K, Schenholm M, Jahnke G, et al. Hyaluronate binding to intact comessas and cultured endothelial cell [J]. Ivest Ophthal Visual Sci, 1989, 30: 2132-2137
[10].Pape LG, Balazs EA. The use of sodium hyaluronate in human anterior segment surgery[J].Ophthalmology, 1980, 87: 699
[11].AIgvere P. Intravitreal implantation of a high-molecular hyaluronic acid in surgery for retinal detachment[J]. Acta Ophthalmol, 1971,49: 975
[12].Bertchez SF, Camber O, Tabatabay C, et al. Use of hyaluronic acid in ocular therapy[A]. In:Edman P ed. Biopharmaceutics of Ocular Drug Delivery[M]. Boca Raton: CRC, 1993:105-120.
[13]. Forrester JV, Balazs EA. Inhibition of phagocytosis by high molecular weight hyaluronate[J].Immunology, 1980,40:435- 446
[14].Dahl LB, Dahl IM, Engstrom-Laurent A, et al. Concentration and molecular weight of sodium hyaluronate in synovial fluids from patients with rheumatoid arthritis and other arthropathies[J].Ann Rheum Dis., 1985, 44: 817-822
[15].Namiki O, Toyoshima H, Morisaki N. High molecular hyaluronic acid[J]. Immunology, 1982,20:50
[16]凌沛学.透明质酸[M].北京:中国轻工业出版社, 2000,53-63
[17].Chung JH, Kim HJ, Fagerholmb P, et al. Effect of topically applied Na-hyaluronan on experimental corneal alkali wound healing[J]. Immunology, 1980, 40: 435- 446
[18].Slevin M, Gaffney J, Kumar S. Signal transduction pathways in hyaluronan induced proliferation of endothelial cells[C]. J Rheumatol, 1993, 39: 13-19
[19].DeAngelis PL, Papaconstantinou J, Weigel PH. Molecular cloning, identification , and sequence of the hyaluronan synthase gene from group A Streptococcus pyogenes[J].Biol.Chem., 1993,268:19181-19184
[20].DeAngelis PL. Molecular directionality of polysaccharide polymerization by the Pasteurella multocida hyaluronan synthase[J]. Biol. Chem., 1999, 274: 26557-26562
[21].Dryden SC, Dowhan W. Isolation and Expression of the Rhodobacter sphaeroides Gene (pgsA) Encoding Phosphatidylglycerophosphate Synthase[J].J Bacteriology,1996,178(4):1030-1038
[22].Weigel PH,Kyossev Z,Torres LC.Phospholipid Dependence and Liposome Reconstitution of Purified Hyaluronan Synthase[J].Biol Chem,2006,281(48):36542-36551
[23].郝宁,张晋宇,陈国强.在兽疫链球菌中表达vgb基因和HA合成基因提高透明质酸产量[J].中国生物工程杂志,2005,25:56-60
[24].Usui M,Sembongi H,Matsuzaki H,et al.Primary Structures of the Wild-Type and Mutant Alleles Encoding the Phosphatidylglycerophosphate Synthase of Escherichia coli[J].J Bacteriology,1994,176:3389-3392
[25].Tlapak-simmons VL,Kempner ES,BaggenstosS BA,et al.The Active Streptococcal Hyaluronan Synthases(HASs)Contain a Single HAS Monomer and Multiple Cardiolipin Molecules[J].J Biol Chem,1998,273:26100-26109
[26].Weigel PH,Kyossev Z,Torres LC.Phospholipid Dependence and Liposome Reconstitution of Purified Hyaluronan Synthase[J].J Biol Chem,2006,281(48):36542-36551
[27].Tlapak-simmons VL,Baggenstoss BA,Kumarl K,et al.Kinetic Characterization of the Recombinant Hyaluronan Synthases from Streptococcus pyogenes and Streptococcus equisimilis[J].J Biol Chem,1999,274(7):4246-4253
[28].Gopalakrishnan AS,Chen YC,Temkin m,et al.Structure and Expression of the Gene Locus Encoding the Phosphatidylglycerophosphate Synthase of Escherichia Coli[J].J Biol Chem,1986,261:1329-1338
[29].Bairoch A.PROSITE:a dictionary of sites and patterns in proteins[J].Nucleic Acids Res,1992,20:2013-2018
[30].Dryden SC,Dowhan W.Isolation and Expression of the Rhodobacter sphaeroides Gene(pgsA)Encoding Phosphatidylglycerophosphate Synthase[J].J Bacteriology,1996,178(4):1030-1038
[2]. Scott JE, Heatley F. Hyaluronan forms specific stable tertiary structures in aqueous solution:a 13C NMR study[J]. Nature Rev. Cancer., 2004,4: 528-539
[3]. Balazs EA. Ultrapure hyaluronic acid and the use thereof[P]. U.S. Patent, 4141973. 1979
[4]. Fraser JR, Laurent TC. Turnover and metabolism of hyaluronan[J]. Ciba Found. Symp., 1989,143:41-53
[5]. Scott JE, Cummings C, Brass A, Chen Y. Secondary and tertiary structures of hyaluronan in aqueous solution,investigated by rotary shadowing-electron microscopy and computer simulation[J]. Biochem., 1991, 274: 699-705
[6]. Prehm P. Hyaluronate is synthesized at plasma mernbranes[J]. Biochem., 1984, 220: 597-600
[7]. Lesley J, Howes N, Perschl A, Hyman R. Hyaluronan binding function of CD44 is transiently activated on T cells during an in vivo immune response[J]. Biochem., 1983, 211: 181 -189
[8]. Balazs EA, Denlinger JL. Viscosupple mentation: A new concept in the treatment of osteoarthritis[J]. J Rheumatol, 1993, 39: 3-9
[9]. Madsen K, Schenholm M, Jahnke G, et al. Hyaluronate binding to intact comessas and cultured endothelial cell [J]. Ivest Ophthal Visual Sci, 1989, 30: 2132-2137
[10].Pape LG, Balazs EA. The use of sodium hyaluronate in human anterior segment surgery[J].Ophthalmology, 1980, 87: 699
[11].AIgvere P. Intravitreal implantation of a high-molecular hyaluronic acid in surgery for retinal detachment[J]. Acta Ophthalmol, 1971,49: 975
[12].Bertchez SF, Camber O, Tabatabay C, et al. Use of hyaluronic acid in ocular therapy[A]. In:Edman P ed. Biopharmaceutics of Ocular Drug Delivery[M]. Boca Raton: CRC, 1993:105-120.
[13]. Forrester JV, Balazs EA. Inhibition of phagocytosis by high molecular weight hyaluronate[J].Immunology, 1980,40:435- 446
[14].Dahl LB, Dahl IM, Engstrom-Laurent A, et al. Concentration and molecular weight of sodium hyaluronate in synovial fluids from patients with rheumatoid arthritis and other arthropathies[J].Ann Rheum Dis., 1985, 44: 817-822
[15].Namiki O, Toyoshima H, Morisaki N. High molecular hyaluronic acid[J]. Immunology, 1982,20:50
[16]凌沛学.透明质酸[M].北京:中国轻工业出版社, 2000,53-63
[17].Chung JH, Kim HJ, Fagerholmb P, et al. Effect of topically applied Na-hyaluronan on experimental corneal alkali wound healing[J]. Immunology, 1980, 40: 435- 446
[18].Slevin M, Gaffney J, Kumar S. Signal transduction pathways in hyaluronan induced proliferation of endothelial cells[C]. J Rheumatol, 1993, 39: 13-19
[19].DeAngelis PL, Papaconstantinou J, Weigel PH. Molecular cloning, identification , and sequence of the hyaluronan synthase gene from group A Streptococcus pyogenes[J].Biol.Chem., 1993,268:19181-19184
[20].DeAngelis PL. Molecular directionality of polysaccharide polymerization by the Pasteurella multocida hyaluronan synthase[J]. Biol. Chem., 1999, 274: 26557-26562
[21].Dryden SC, Dowhan W. Isolation and Expression of the Rhodobacter sphaeroides Gene (pgsA) Encoding Phosphatidylglycerophosphate Synthase[J].J Bacteriology,1996,178(4):1030-1038
[22].Weigel PH,Kyossev Z,Torres LC.Phospholipid Dependence and Liposome Reconstitution of Purified Hyaluronan Synthase[J].Biol Chem,2006,281(48):36542-36551
[23].郝宁,张晋宇,陈国强.在兽疫链球菌中表达vgb基因和HA合成基因提高透明质酸产量[J].中国生物工程杂志,2005,25:56-60
[24].Usui M,Sembongi H,Matsuzaki H,et al.Primary Structures of the Wild-Type and Mutant Alleles Encoding the Phosphatidylglycerophosphate Synthase of Escherichia coli[J].J Bacteriology,1994,176:3389-3392
[25].Tlapak-simmons VL,Kempner ES,BaggenstosS BA,et al.The Active Streptococcal Hyaluronan Synthases(HASs)Contain a Single HAS Monomer and Multiple Cardiolipin Molecules[J].J Biol Chem,1998,273:26100-26109
[26].Weigel PH,Kyossev Z,Torres LC.Phospholipid Dependence and Liposome Reconstitution of Purified Hyaluronan Synthase[J].J Biol Chem,2006,281(48):36542-36551
[27].Tlapak-simmons VL,Baggenstoss BA,Kumarl K,et al.Kinetic Characterization of the Recombinant Hyaluronan Synthases from Streptococcus pyogenes and Streptococcus equisimilis[J].J Biol Chem,1999,274(7):4246-4253
[28].Gopalakrishnan AS,Chen YC,Temkin m,et al.Structure and Expression of the Gene Locus Encoding the Phosphatidylglycerophosphate Synthase of Escherichia Coli[J].J Biol Chem,1986,261:1329-1338
[29].Bairoch A.PROSITE:a dictionary of sites and patterns in proteins[J].Nucleic Acids Res,1992,20:2013-2018
[30].Dryden SC,Dowhan W.Isolation and Expression of the Rhodobacter sphaeroides Gene(pgsA)Encoding Phosphatidylglycerophosphate Synthase[J].J Bacteriology,1996,178(4):1030-1038