新型牛溶菌酶基因的重组真核质粒构建及活性研究
|
摘要
溶菌酶(lysozyme，LYZ)是一种专门作用于微生物细胞壁的水解细菌细胞壁中粘多糖β-1，4-糖苷键的胞壁质酶，是一种碱性蛋白质，存在于许多动物、植物和微生物中。研究发现，溶菌酶对革兰氏阳性细菌具有直接的溶解作用，催化革兰氏阳性菌细胞壁不溶性多糖水解，生成可溶性黏肽。在分泌型免疫球蛋白A和补体的参与下，溶菌酶还对革兰氏阴性细菌具有间接的溶解作用，是一种天然的抗菌物质。由于其能够增强巨噬细胞和白细胞的吞噬和消化功能、将诱发炎症的酸性物质灭活、增强抗菌素及其它药物的疗效，具有消炎消肿、帮助组织修复、改善组织局部血液循环和分解脓液等药理作用，因此溶菌酶在医疗界中倍受关注。近年来，随着生物学技术和免疫学技术的发展，对溶菌酶的研究取得了令人瞩目的成就。利用DNA重组技术发展溶菌酶的抗菌效果，已成为其中重要的研究方向。
牛体内溶菌酶(bovine lysozyme，bLys)有大约10种，表达的潜在位点有：乳腺、唾液、泪液、胃、气管组织以及中性粒细胞、嗜红细胞、巨噬细胞、白细胞等。由于牛奶与人们的生活息息相关，其质量的好坏关系着人们的健康，因此，对牛奶溶菌酶的研究尤为突出。为构建具有高效抗菌、活性持久的抗菌基因，探索研制新型抗菌制剂，克服耐药性细菌感染日益严重的问题，本实验构建了原核表达载体pGEX-4T-1-Lys(pGL)。本实验是以成功构建的新型牛溶菌酶基因原核表达载体pGL为模板，用PCR扩增得到目的片段Lys基因，将VR1020和目的片段Lys分别用BamHI和BglⅡ双酶切，并将VR1020酶切片段先进行去磷酸化，然后将两种酶切产物在16℃水浴过夜连接；连接产物转化大肠杆菌DH5α感受态细胞，筛选重组子，用碱裂解法小规模提取重组子质粒DNA，用BamHI，BglⅡ作双酶切鉴定验证，并作质粒PCR鉴定。经双酶切和质粒PCR的实验结果初步确定的阳性重组子进行双向基因测序，测序结果证明插入目的片段正确，证实新型牛溶菌酶基因已经克隆到真核表达载体VR1020(VR1020-Lys，命名为VRL)上。
将重组质粒VRL以JetPEI~(TM) Cationic polymer分子包裹，介导转染真核HEK293细胞，转染培养24h、48h、72h后分别提取培养细胞mRNA进行RT-PCR，与VR1020转染细胞比较发现，转染后24h、48h和72h的HEK293细胞的RNA中含有与目的cDNA片段对应的mRNA。为了探索一种更经济高效的转染介质，本实验同时还制备了壳聚糖纳米颗粒(chitosan nanoparticles，CNP)，用此壳聚糖纳米颗粒包裹重组质粒VRL后转染HEK293细胞，转染的HEK293细胞中也表达与目的cDNA片段对应的mRNA，说明了壳聚糖纳米颗粒也能作为一种有效的转染介质。用转染后HEK293细胞上清分别作抑制标准大肠杆菌、产毒性大肠杆菌、绿脓杆菌、金黄色葡萄球菌和肺炎球菌生长实验，实验结果证明新构建的真核表达质粒VRL具有良好的抑制革兰氏阳性菌生长的效果，这些为深入开展VRL在动物体内的抗感染抑菌实验奠定了可靠的基础。
Lysozyme, which was recognized as bacteriolytic agent having an ability to hydrolyze bacterial cell walls, is found in a variety of vertebrate cells and secretions, such as spleen, milk, tears and egg white. It lyses certain bacteria by hydrolysing theβ-linkages between the muramic acid and N-acetylglucosamine of the mucopolysaccharides which are present in the bacterial cell wall. It has become one of the most intensively studied proteins. The action of Lysozyme on bacteria works cooperatively and synergistically with antibiotics, which has a very important practical value in medicine area. In present, the research of lysozyme develops greatly because of the development of the technology of biology and immunology. And it becomes very import that using the technology of DNA recombination to boost the antibacterial effect of lysozyme.
There are about ten kinds of bovine lysozyme that are potentially produceded in neutrophil and macrophage in mammary gland, stomach and tracheal tissue, and secreted into saliva, tears fluid and milk which is an important food for human beings. In order to construct a novel lysozyme gene to improve its antibacterial activity and develop effective medicine to overcome the increasing infection of animals by antibiotic resistant bacteria, the experiment was carried out to reconstruct the eukaryotic expression vector for bovine milk lysozyme gene (Lys) by inserting the
Lys cDNA fragment into VR1020 plasmid. The VR1020 was digested by BamHⅠand BglⅡ, and then ligated with Lys cDNA digested wit hthe same double endoenzymes at 16℃overnight. The ligated products were used to transform E. coli DH5αcompetent cell, and the positive recombinant was screend out by inoculation of transformed cells in Lurea Bertani (LB) broth with kanamycin at 100μg/ml and shaked for 16 hours at 37℃. Bacterial cells were pelleted by centrifugation and plasmid DNA extracted following large-scale alkine lysis. Then the recombinant plasmid was digested by BamHⅠand BglⅡand plasmid PCR to identify the cloned Lys gene. Through DNA sequencing, it was proved that the Lys cDNA fragment was successfully inserted into the eukaryotic plasmid VR1020 in correct orientation.
The ability of VRL to express Lys in vitro was assayed by RT-PCR in eukaryotic cells HEK293 that were transiently transfected with VRL wraped in JetPEI~(TM) Cationic polymer transfection reagent and chitosan nanoparticles (CNP). The result was found that the mRNA was detected in the the total RNA of HK293 cells transfected after 24, 48, 72h; It was also showed that the expression effect of the gene enwrapped by CNP was the same with or better than that of JetPEI~(TM) Cationic polymer, indicating CNP is practicable package to facilitate the plasmid transfectection of eukaryotic cells. The expression products in the supernatant of transfected cell medium manifested significant inhibitary effect to suppress the growth of Streptococcus pneumoniae and Staphylococcus aureus bacteria in vitro(P＜0.05), and this suggest that the recombinant eukaryotic plasmids of novel bovine lysozyme gene could be further developed into effective antibacterial reagent to control animal infection.
牛体内溶菌酶(bovine lysozyme，bLys)有大约10种，表达的潜在位点有：乳腺、唾液、泪液、胃、气管组织以及中性粒细胞、嗜红细胞、巨噬细胞、白细胞等。由于牛奶与人们的生活息息相关，其质量的好坏关系着人们的健康，因此，对牛奶溶菌酶的研究尤为突出。为构建具有高效抗菌、活性持久的抗菌基因，探索研制新型抗菌制剂，克服耐药性细菌感染日益严重的问题，本实验构建了原核表达载体pGEX-4T-1-Lys(pGL)。本实验是以成功构建的新型牛溶菌酶基因原核表达载体pGL为模板，用PCR扩增得到目的片段Lys基因，将VR1020和目的片段Lys分别用BamHI和BglⅡ双酶切，并将VR1020酶切片段先进行去磷酸化，然后将两种酶切产物在16℃水浴过夜连接；连接产物转化大肠杆菌DH5α感受态细胞，筛选重组子，用碱裂解法小规模提取重组子质粒DNA，用BamHI，BglⅡ作双酶切鉴定验证，并作质粒PCR鉴定。经双酶切和质粒PCR的实验结果初步确定的阳性重组子进行双向基因测序，测序结果证明插入目的片段正确，证实新型牛溶菌酶基因已经克隆到真核表达载体VR1020(VR1020-Lys，命名为VRL)上。
将重组质粒VRL以JetPEI~(TM) Cationic polymer分子包裹，介导转染真核HEK293细胞，转染培养24h、48h、72h后分别提取培养细胞mRNA进行RT-PCR，与VR1020转染细胞比较发现，转染后24h、48h和72h的HEK293细胞的RNA中含有与目的cDNA片段对应的mRNA。为了探索一种更经济高效的转染介质，本实验同时还制备了壳聚糖纳米颗粒(chitosan nanoparticles，CNP)，用此壳聚糖纳米颗粒包裹重组质粒VRL后转染HEK293细胞，转染的HEK293细胞中也表达与目的cDNA片段对应的mRNA，说明了壳聚糖纳米颗粒也能作为一种有效的转染介质。用转染后HEK293细胞上清分别作抑制标准大肠杆菌、产毒性大肠杆菌、绿脓杆菌、金黄色葡萄球菌和肺炎球菌生长实验，实验结果证明新构建的真核表达质粒VRL具有良好的抑制革兰氏阳性菌生长的效果，这些为深入开展VRL在动物体内的抗感染抑菌实验奠定了可靠的基础。
Lysozyme, which was recognized as bacteriolytic agent having an ability to hydrolyze bacterial cell walls, is found in a variety of vertebrate cells and secretions, such as spleen, milk, tears and egg white. It lyses certain bacteria by hydrolysing theβ-linkages between the muramic acid and N-acetylglucosamine of the mucopolysaccharides which are present in the bacterial cell wall. It has become one of the most intensively studied proteins. The action of Lysozyme on bacteria works cooperatively and synergistically with antibiotics, which has a very important practical value in medicine area. In present, the research of lysozyme develops greatly because of the development of the technology of biology and immunology. And it becomes very import that using the technology of DNA recombination to boost the antibacterial effect of lysozyme.
There are about ten kinds of bovine lysozyme that are potentially produceded in neutrophil and macrophage in mammary gland, stomach and tracheal tissue, and secreted into saliva, tears fluid and milk which is an important food for human beings. In order to construct a novel lysozyme gene to improve its antibacterial activity and develop effective medicine to overcome the increasing infection of animals by antibiotic resistant bacteria, the experiment was carried out to reconstruct the eukaryotic expression vector for bovine milk lysozyme gene (Lys) by inserting the
Lys cDNA fragment into VR1020 plasmid. The VR1020 was digested by BamHⅠand BglⅡ, and then ligated with Lys cDNA digested wit hthe same double endoenzymes at 16℃overnight. The ligated products were used to transform E. coli DH5αcompetent cell, and the positive recombinant was screend out by inoculation of transformed cells in Lurea Bertani (LB) broth with kanamycin at 100μg/ml and shaked for 16 hours at 37℃. Bacterial cells were pelleted by centrifugation and plasmid DNA extracted following large-scale alkine lysis. Then the recombinant plasmid was digested by BamHⅠand BglⅡand plasmid PCR to identify the cloned Lys gene. Through DNA sequencing, it was proved that the Lys cDNA fragment was successfully inserted into the eukaryotic plasmid VR1020 in correct orientation.
The ability of VRL to express Lys in vitro was assayed by RT-PCR in eukaryotic cells HEK293 that were transiently transfected with VRL wraped in JetPEI~(TM) Cationic polymer transfection reagent and chitosan nanoparticles (CNP). The result was found that the mRNA was detected in the the total RNA of HK293 cells transfected after 24, 48, 72h; It was also showed that the expression effect of the gene enwrapped by CNP was the same with or better than that of JetPEI~(TM) Cationic polymer, indicating CNP is practicable package to facilitate the plasmid transfectection of eukaryotic cells. The expression products in the supernatant of transfected cell medium manifested significant inhibitary effect to suppress the growth of Streptococcus pneumoniae and Staphylococcus aureus bacteria in vitro(P＜0.05), and this suggest that the recombinant eukaryotic plasmids of novel bovine lysozyme gene could be further developed into effective antibacterial reagent to control animal infection.
引文
1. Blackwell JR, Horgan R. A novel strategy for production of a highly expressed recombinant protein in an active form. FEBS Letters, 1991, 295 (1-3): 10-12
2. Bodmeier R, Chert HG, Paeratakul O.A. Novel approach to the delivery of micro or nanoparticles. Pharm Res, 1989,6 (5): 413-417
3. Calvo P, Remunan-Lopez C, Vila-Jato JL, et al. Novel hydrophilic chitosan/polyethylene oxide nanoparticles as proteins carders. Int J Pharm,2001, 224 (1/2): 159-168
4. Chellat F, Tabrizian M, Dumitriu S, et al. In vitro and in vivo biocompatibility of chitosan-xanthan polyionic complex. J Biomed Res, 2000, 51 (1): 107-116
5. Chen J. et al. Enhancement of the solubility of proteins overexpressed in Escherichia coli by heat shock. J. Mol. Microbiol Biotechnol. 2002, 4 (6):519-524
6. David M. Irwin, Allan C. Wilson. Multiple cDNA Sequences and the Evolution of Bovine Stomach Lysozyme. The Journal of biological chemistry, 1989, 264 (19): 11387～11393
7. Derman AI et al. Mutation that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science, 1993,262 (5140): 1744-1747
8. Dinsmore R.E, et al. OxytetracycLane residues in milk in intrauterine infusion of dairy cows. Boy. Proc, 1994, 26:187-289
9. Hain Ruml udiger, Stenzel Klaus. Use of lysozyme gene structures in plants to increase resistance. Biotechmology Advances, 1997, 15 (3-4): 665
10. Hartley DL, et al. Biochemical Society transactions. 1988,16:101
11. Hayakawa T, ToibanaA, Marumoto R etal. Gene, 1987, 66:53-59
12. Hernandez VP. et al. Characterization and cDNA cloning of an immune-induced lysozyme from cultured Aedes albopictus mosquito cells. Developmental and Comparative Immunology, 2003,27 (1): 11-20
13. Hikima Jun-ichi et al. Molecular cloning, expression and evolution of the Japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein. Biochimica et Biophysica Acta (BBA)/Gene Structure and Expression, 2001,1520 (1): 35-44
14. Hikima S. et al. Characterization and flmction of kuruma shrimp lysozyme possessing lytic activity against Vibrio species. Gene, 2003,316:187-195
15. Hideynki A, Hisham RI, Takeshi K, et al. Bactericidal action of lysozymes attached with various sizes of hydrophobic peptides to the C-terminal using genetic modification. FEBS Letters, 1997,415:114-118
16. Hockney, RC. Recent developments in heterologous protein production in E.coli. Trends Biotech. 1995,13:456-463
17. Horton R, Hunt H, Ho S. Engineering hybrid gene without the use of restriction enzymes: gene splicing by overlap extension. Gene, 1989,77:61-68
18. Kazuhiko Takeuchi, David M.Irwin et al. Multiple cDNA Sequences of Bovine Tracheal Lysozyme. The Journal of biological chemistry, 1993,268 (36): 27440-27446
19. Krueger JK, Stock AM, Schutt CE and Stock J.BioPham. In LM Gierasch and J King eds. Protein foIding:Deciphering the second half of the genetic code.American association for the advancement of science, Washington,D.C, 1990.136-142
20. Lavallie ER, DiBlasio EA, Kovacic S. et al. A thioredoxin gene fusion expression system that circumvents inclusion body formatin in the E.coli cytoplasm. Biotechnology, 1993, 11: 187-193
21. Li B, Huang Y, Paskewitz SM. Hen egg white lysozyme as an inhibitor of mushroom tyrosinase. FEBS Lett. 2006,580 (7): 1877-1882
22. Liu Feng, Wen Zilong. Cloning and expression pattern of the lysozyme C gene in zebrafish. Mechanisms of Development, 2002,113(1): 69-72
23. Luo ZH, Hua ZC. Increased solubility of glutathione S-transferase-P16 (GST-p16) fusion protein by co-expression of chaperones groes and groel in Escherichia coli. Biochem. Mol. Biol. Int. 1998,46 (3): 471-477
24. Maga E, Anderson G, Huang M, et al. Expression of human lysozyme mRNA in the mammary gland oftransgenic mice. Transgenic Research, 1994, 3:36-42
25. Mao HQ, Roy K, Troung Le VL, et al. Chitsan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. J Control Release, 2001,70 (3): 399-421
26. Mafia J, Walter S, Gunther R. Cloning of human lysozyme gene and expression in the saccha romyces cerevisiae.Gene, 1988, 66:223-234
27. Maston AO. The purification of eukaryotic polypeptides synthesized in E.coli. Biochem. J, 1986, 240:1-12
28. Moss JM. et aL Purification, Characterization, and Biosynthesis of Bovine Cartilage Lysozyme Isoforms. Archives of Biochemistry and Biophysics, 1997, 339 (1): 172-182
29. Muraki M, Jigami Y, Tanaka H etal. Agric. Biol. Chem. 1986, 60 (3): 713-723
30. Nakimbugwe D. et al. Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts. FEMS Microbiol. Lett, 2006, 259 (1): 41-46
31. Qan SJ, Yu Y, Tiang KQ. Synthesis and cloning of the human lysozyme gene. Sheng wu Gong cheng Xue bao (Chin. J. Bio. Technol.), 1994, 10(1): 34-38
32．Rebhun WC．等著，赵德明等译。《奶牛疾病学》。北京：中国农业大学出版社，1999，427—432
33. Rinas U. et al. Cysteine to serine substitutions in basic fibmblast growth factor effect on inclusion body formation and proteolytic susceptibility during in vitro refolding. Biotechnology(NY), 1992, 10 (4): 435-440
34. Ruyssen R. Pharmaceutical Enzymes, Properties and Assay Methods. E Storyscientia PVBA, Gent 1978
35. RuttloffH. Industrielle Enzvme Steinkopff, Darmstadt 1979
36．Sambrook J，Fritsch EF,Maniatis T.分子克隆实验指南，第2版，北京：科学出版社，1995
37. Schein CH, Noteborn HM. Formation of solutable recombinant proteins in E.coli is favored by lower growth temperature. Biotechnology, 1988, 6:291-294
38. Shigeru M, Masato K, Masanorl N, et al. Secretion of active human lysozyme by Acremonium chrysogenum using a Fusarium alkaLane protease promoter system. Journal of Biotechnology, 1995, (42): 1-8
39. Tsuchiya K, Tada S, Gomi K et al. App[. Microbiol. Biotechnol. 1992, 38:109-114
40. Usui M. et al. Effective reduction of antigenicity of hen egg lysozyme by site-specific glycosylation. FEBS Letters, 2004, 557 (1-3): 169-173
41. Wang Dianliang. Research Progress in Recombinant Human Lysozyme. China Biotechnology, 2003, 23 (9): 59-63
42. Wen Yi, Irwin David M. Mosaic Evolution of Ruminant Stomach Lysozyme Genes. Molecular Phylogenetics and Evolution, 1999, 13 (3): 474-482
43. Xie Y, Wetlaufer DB. Control of aggregation in protein refolding: the temperature-leap tactic. Prorein Sci, 1996, 5 (3): 517-523
44. Yumiko Shirano et al. Cultivation of E.coli carryinga rice lipoxygenase L-2cDNA produce a soluble and active enzyme at a high level. FEBS Letters, 1990, 271: 128-130
45. Zhang Chun-yan, Ning Bo, Cao Kai-yuan. Cloning ofhCD154 gene from human activated peripheral blood mononuclear cell and expression of hCD154-GST fusion protein in prokaryote. Chinese Journal Of Pathophysiology, 2000, 16 (8): 673-677
46. Zhang Yi-Zheng. Basic experiments in molecular cloning, 1995,11
47．曹访，唐修君，陈丽，王杏龙。奶牛乳腺炎研究进展，黑龙江动物繁殖，2006年，第14卷，第4期，13-15
48．高焕春，吕晓玲，李文英。鸡蛋清溶菌酶提取工艺及其应用初探，天津轻工业学院学报，1996，(1)：37-40
49．胡士明。对奶牛乳房炎发病机制的新认识及临床处理对策的探讨，中国奶牛，1998： 41-44
50．胡松华，杜爱芳，蔡渭明。奶牛临床型和隐性型乳房炎的细菌学分析，中国畜禽传染病，1998，20(4)：199-201
51．贾向志，李元，马文煜。溶菌酶的研究进展，生物技术通讯，2002，13(5)，374-377
52．矫庆华，钱世均，叶军等。人工合成人溶菌酶基因在大肠杆菌中表达，生物工程学报，1997，13(1)：102—104
53．林锋强，潘杭君，胡松华。奶牛乳房炎疫苗研究进展，中国奶牛，2002，1：40—42
54．林向阳，何承云，阮榕生，刘玉环，郑丹丹，朱榕璧。溶菌酶及其应用研究，中国食 品添加剂，2005，No6，105-106
55．李明，孙彦伟。奶牛乳腺炎的防治与研究动态，广东畜牧兽医科技，2006，3，11-14
56．刘宝良，黄俊武。乳房炎的发病原因与自身防御能力，畜牧兽医科技信息，2005，4：67
57．刘玉清，唐兆新。奶牛乳腺炎防治的研究现状，现代畜牧兽医，2006，1，48-50
58．吕俊。溶菌酶治疗子宫内膜炎的试验情况。上海奶牛，2000，24(2)：42—43
59．母安雄，胡松华。奶牛乳房炎抗生素防治失败原因探讨，中国兽医杂志，2002，2：18—20
60．荣晓花，凌沛学。溶菌酶的研究进展，中国生化药物杂志，1999，20(6)：319—320
61．孙怀昌。人溶菌酶基因治疗奶牛乳腺炎的初步研究，畜牧兽医学报，2004，35(2)：227—232
62．孙怀昌。防治奶牛乳房炎的基因工程新药，河北畜牧兽医，2005年第21卷第10期，12-13
63．王慎行。奶牛隐性乳房炎调查及纯中药“消炎增乳灵”治疗试验，中兽医医药杂志，2006，2：38—40
64．谢磊。大肠杆菌表达系统及其研究进展，华南热带农业大学学报，2004，10(2)：16—20
65．杨龙骐，沈永恕，彭义。奶牛乳房炎防治研究进展，郑州牧业工程高等专科学校学报，2001，21(2)：178—181
66．周宇荀，魏东芝。融合蛋白表达载体pGEX及其应用，生命科学，1998，10(3)：122—124
1. Adriana I, Licz A, Laura U, et al. Determination of the fractal dimension of the lysozyme backbone of three diferent organisms, Chaos, Solitons and Fractals, 2001, (12): 757—760
2. Audy P, Quere DL, Leclerc D, et al. Electrophoretic forms of lysozyme activity in various plant species. Phytochemistry, 1990, 29 (4) :1143～1159
3. Bae Sangki, Kim Yonggyun. Lysozyme of the beet arrnyworm, Spodoptera exigua: activity induction and cDNA structure. Comparative Biochemistry and Physiology, Part B, 2003, 135(3): 511-519
4. Bomimcomtro A., Onori G. Investigation by dielectric spectroscopy of domain motions in lysozyme: effect of solvent and binding of inhibitors, Chemical Physics Letters, 2004, 398(1-3): 260-263
5. Caballero M, Ruiz R, Marquez M, et al. Development of a Micmparticle-Enhanced Nephelometric Immunoassay for Quantitation of Human Lysozyme in Pleural Efusion and Plasma, Journal of Clinical Laboratory Analysis, 1999, (13): 301-307
6. Castanon M J, Spevak W, A do lf G R, et al. Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae [J], Gene, 1988,66: 223-234.
7. Chipman DM, Sharon N. Mechanism of lysozyme action. Science, 1969,165:454
8. Conte A, Buonocore GG, Bevilacqua A, Sinigaglia M, Del Nobile MA. Immobilization of lysozyme on polyvinylalcohol films for active packaging applications. J, Food Prot, 2006,69 (4): 866-870
9. Chung L P, Keshav S, Go rdon S, et al. Cloning the human lysozyme cDNA: inverted A lurepeat in the mRNA and in situ hybridization for macrophages and Paneth cell [J], P rocN atlAcad Sci USA, 1988, 85 (17): 6 227-6 231.
10. Diels Ann MJ, et al. Sensitisation of Escherichia coli to antibacterial peptides and enzymes by high-pressure homogenization, International Journal of Food Microbiology, 2005,105 (2): 165-175
11. Durance T D, Nakai S. Simultaneous isolation of avidin and lysozyme from egg albumen [J], Journal of food science, 1988,53 (4): 1096
12. Ghosh R, Cui ZF. Protein purification by ultrafiltration with pretreated membrace [J],Journal of membrane science, 2000,167:47
13. Glazer AN, Barel AO, Howard JB, et al. Isolation and characterization of fig lysozyme. J., Biol. Chem., 1969,244 (13): 3583-3589
14. Hain Ruml udiger, Stenzel Klaus. Use of lysozyme gene structures in plants to increase resistance, Biotechmology Advances, 1997,15 (3-4): 665
15. He LZ, Sun Y. Purification of lysozyme by multistage affinity filtration [J], Bioprocess BiosystEng, 2002,25:155
16. Hernandez VP, et al. Characterization and cDNA cloning of an immune-induced lysozyme from cultured Aedes albopictus mosquito cells, Developmental and Comparative Immunology, 2003,27 (1): 11-20
17. Hikima Jun-ichi et al. Molecular cloning, expression and evolution of the Japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein, Biochimica et BiophysicaActa (BBA)/Gene Structure and Expression, 2001,1520 (1): 35-44
18. Hikima S. et al. Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species, Gene, 2003,316:187-195
19. Howard JB, Giazer AN. Studies of the physicochemical and enzymatic properties of papaya lysozyme. J, Biol. Chem., 1967,242 (24): 5751-5723
20. Huang B, Zhao C, Lei X, et al. The cloning, sequencing and analysis of Chinese human lysozyme gene. cDNA amplified with RT-PCR from human placental total RNA [J ], Chin BiochemJ, 1993,9:2692273.
21. Ibrahim HR. et al. Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function, FEBS Letters. 2001,506 (1): 27-32
22. Ivaanov AE, Galaev IY, Kazakov SV, et al. Thermosensitive copolymers of N-Vinylimidazole as displacers of proteins in immobilized metal aaffinity chromatography [J], Journal of chromatohraphy A, 2001, 907:115
23. Jolles J, Jolles P. Human milk lysozyme: unpublished data concerning the establishment of the complete primary structure; comparison with lysozymes of various o rigins [J], Helv Chim Acta, 1971, 54 (8): 2 668-2 675.
24. Jonathan M. Moss, et.al. Purification, Characterization, and Biosynthesis of Bovine Cartilage Lysozyme Isoforms, Archives of Biochemistry and Biophysics, 1997,339 (1): 172-182
25. Kang HK, Chung YJ, Park CU, Jang YS, Kim BS. Induction of autoimmunity by immunization with hapten-modified hen egg lysozyme in hen egg lysozyme-transgenic mice., Immunology, 2006, 117 (3): 368-378
26. Leheninger AL, Nelson DL, Cox MM. Principles of Biochemistry, 2nd Edition. New York: Worth Publishers, 1993: 180,312
27. Li B, Huang Y, Paskewitz SM. Hen egg white lysozyme as an inhibitor of mushroom tyrosinase, FEBS Lett, 2006, 580 (7): 1877-1882
28. Li chang E, Nakai S, Sim J, et al. Lysozyme sepratation from egg white by cation exchang column chromatography[J], Journal of food science, 1986,51 (4):1 032
29. Liu Feng, Wen Zilong. Cloning and expression pattern of the lysozyme C gene in zebrafish, Mechanisms of Development, 2002,113(1): 69-72
30. Lockey TD, Ourth DD. Purification and Characterization of Lysozyme from Hemolymph of Heliothis virescens Larvae, Biochemical and Biophysical Research Communications, 1996,220 (3): 502-508
31. Lopez-Jaramillo FJ, Perez-Banderas F, Hernandez-Mateo F, Santoyo-Gonzalez F, Production, crystallization and X-ray characterization of chemically glycosylated hen egg-white lysozyme, Acta Crystallograph Sect F Struct Biol Cryst Commun. 2005,61 (Pt 4): 435-438
32. Lord MS. et al. Lysozyme interaction with poly (HEMA)-based hydrogel, Biomaterials, 2006,27(8): 1341-1345
33. Lynn KR. Chitinases and chitobioased form the American lobster. Homarus. Biochem. Physiol., 1990, (96B): 761-766
34. Marcano JG, Tsotisis TT. Catalytic membranes and membrane reactors, Wiley-VCH Verlag GmbH, Weinheim Press, 2002
35. Marc Henke, Gerd Hobom, et.al. Strucral deviations in a bovine low expression lysozyme-ecoding gene active in tissues other than stomach, Gene, 1996,178:131-137
36. Moss JM. et al. Purification, Characterization, and Biosynthesis of Bovine Cartilage Lysozyme Isoforms, Archives of Biochemistry and Biophysics, 1997,339 (1): 172-182
37. Mudgil P, Torres M, Millar TJ. Adsorption of lysozyme to phospholipid and meibomian lipid monolayer films, Colloids Surf B Biointerfaces, 2006,48(2): 128-137
38. Murakand M, et al. Expression of synthetic human lysozyme gene in Escherichia coli, Agricultural Biological Chemistry, 1986,50: 713-723
39. Nattress FM. et al. Evaluation of the ability of lysozyme and nisin to control meat spoilage bacteria, International Jourmal of Food Microbiology, 2001,70 (1-2): 111-119
40. Ohkuri T. et al. Effect of the Structure of the Denatured State of Lysozyme on the Aggregation Reaction at the Early Stages of Folding from the Reduced Form, Journal of Molecular Biology, 2005,347 (1): 159-168
41. Perry LJ, et al. Disulfide bond engineered into T4 lysozyme: stabilization of the protein toward thermal inactivation, Science, 1984,226:555-557
42. Sasagawa N, Saito K, Sugita K. Ionic cross-linking of SO_3H-groupcontaing graft chains helps to capture lysozyme in a permeation mode [J], Journal of chromatography, 1999,848 (1):161
43. Serafica GC, Pimbley J, Belfon G. Protein fraction using fast flow immobilized metal chelate affinity membranes [J], Biotech and Bioeng, 1994,43:21
44. Shireru M, et al. Secretion of active human lysozyme by Acremonium chrysogenum using fusarium alkaLane protease system, Journal of Biotechnology, 1995,45:1-8
45. Sugahara T. et al. Heat denaturation enhanced immunoglobulin production stimulating activity of lysozyme from hen egg white, Biochimica et Biophysica Acta (BBA)/General Subjects, 2002, 1572(1): 19-24
46. Sun HC, Xue FM, Qian K, Fang HX, Qiu HL, Zhang XY, Yin ZH. Intramammary expression and therapeutic effect of a human lysozyme-expressing vector for treating bovine mastitis. J, Zhejiang Univ Sci B, 2006, 7 (4): 324-330
47. Tata SJ, Bemtema JJ, Balabaskaram S. The lysozyme of Hevea brasiliensis latex: isolation, purification, enzymekinetics and a partial amino-acid sequence. J, Rubber Res. Malaya, 1983,31(1): 35-48
48. Usui M. et al. Effective reduction of antigenicity of hen egg lysozyme by site-specific glycosylation, FEBS Letters, 2004,557 (1-3): 169-173
49. Vaidya AA, Lele BS, Kulkarmi MG, et al. Thermmoprecipition of lysozyme from egg white using copolymers of N-isopropylaacrylaamide and acidic monomers[J], Journal of biotechnology, 2001,87:98
50. Vaidya AA, Lele BS, Deshmukh MV, et al. Design and evaluation of new ligands for lysozyme recovery by affinity thermoprecipitation[J], Chemical Engineering Science 2001, 56:5681
51. Vieira MN, Figueroa-Villar JD, Meirelles MN, Ferreira SR, De Felice FG Small Molecule Inhibitors of Lysozyme Amyloid Aggregation, Cell Biochem. Biophys, 2006, 44 (3): 549-553
52. Wen Yi, Irwin David M. Mosaic Evolution of Ruminant Stomach Lysozyme Genes, Molecular Phylogenetics and Evolution, 1999, 13 (3): 474-482
53. Yakobi M, et al. Purifieation and characterization of chiltinase and chitobiase produced by Acromonas hydrophila sup sp, Anaerogenes A52. J. Gen. Appl, Microbiol, Tokyo, 1986, (32): 2538
54. Yoshimura K, Toibana A, N akahama K. Human lysozyme: sequencing of a cDNA, and expression and secretion by Saccharomyces cerevisiae [J], Biochem Biophys Res Commun, 1988, 150(2): 794-801.
55. Youtao Song, et al. In vivo glycosylation suppresses the aggration ofamyloidogenic hen egg white lysozymes esoressed in yeast, FEBS Letters, 2001, 491:63-66
56. Yu Mei, Irwin DM. Evolution of Stomach Lysozyme: The Pig Lysozyme Gene, Molecular Ohylogenetics and Evolution, 1996, 5 (2): 298-308
57. Zhang G, Darius S, Smith SR, Ritchie SJ. In vitro inhibitory effect of hen egg white lysozyme on Clostridium perfringens type A associated with broiler necrotic enteritis and its alpha-toxin production, Lett Appl Microbiol, 2006, 42(2): 138-143
58. Zhang Xing, Studier FW. Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme., Journal of Molecular Biology, 1997, 269 (1): 10-27
59．苍金荣等。微量快速比浊法检测溶菌酶含量，陕西医学检验，1994，9(1)：38—39
60．柴红，陈欢林，刘茉娥。固定金属离子亲和膜分离技术(Ⅱ)研究进展和应用前景[J]，膜科学与技术，1999，19(5)：13
61．陈慧英，吴晓英，林影。溶菌酶分离纯化方法的研究新进展，广东药学院学报，2003，19(4)：355—258
62．船津胜，鹤大典。溶菌酶，济南：山东科技出版社，1982：39～78，257～262
63．戴清源，陈祥贵，李晓霞，张庆。溶菌酶的研究进展，内蒙古农业科技2005(3)：14～16
64．冯惠勇，徐亲民，孙国志。溶菌酶的生物测定方法研究，食品与发酵工业，2002，28(4)：60-64
65．凤权，汤斌。溶菌酶分离纯化方法的研究进展，生物学杂志，2006年2月第23卷第一期，5—7
66．高焕春，吕晓玲，李文英。鸡蛋清溶菌酶提取工艺及其应用初探，天津轻工业学院学报，1996，(1)：37—40
67．高向阳，袁四清。萝卜溶菌酶抑菌效果的初步研究，华南农业大学学报，1997，2：72—75
68．郭新竹。天然肽类防腐剂研究进展，食品与发酵工业，2000，27(2)：72—75
69．郭勇。酶的生产与应用，北京：化学工业出版社，2003，98—101
70．贾向志，袁汉英，马文煜，李元，李育阳。人溶菌酶基因的克隆及其在毕赤酵母中的表达，第四军医大学学报2001；22(22)，2068—2072
71．姜秀云，吴惠毅。平板法测定血清溶菌酶活性影响因素探讨上海医学检验杂志，1994，9(1)：12-13
72．矫庆华等。人工合成人溶菌酶基因在大肠杆菌中表达，生物工程学报，1997，13(1)：102-104
73．柯从玉，白泉，耿信笃。用于变性蛋白质复性及同时纯化的简易型色谱柱的研制及其应用，色谱，2004，22 (4)：394—398
74．李春联。微量溶菌酶比色测定法在临床诊断中的应用，同济大学学报(医学版)，2002，6 (3)：251
75．李国才，孙怀昌，钱科，居杏珠，陈瑾，张泉，朱秋菊。重组人溶菌酶理化及生物学特性的鉴定，中国乳品工业，2005年，Vol 33，No 4，11—14
76．李静，陈欢林，柴红。金属螯合亲和膜吸附分离与纯化溶菌酶的研究，膜科学与技术，2001，21 (4)：5—9
77．林翠花，肖素荣，孟庆国。溶菌酶结构特点及其应用，潍坊学院学报，2005，5 (2)：108-110
78．刘梅，张士璀。溶菌酶C及其生物学功能，生命的化学，2006年26卷5期，465—467
79．刘世清。牛乳中溶菌酶的生物学特性及其应用，畜牧兽医科技信息，2005，4：66—67
80．刘莹，孙荣丹，杨翔，华王丽。溶菌酶的应用现状，饲料工业，2006年第27卷第6期，19—20
81．楼善贤，苗德林。溶菌酶的研究进展，中国肿瘤临床，1994，21(9)：709—711
82．马绪荣，苏德模主编。药品微生物学检验手册，北京：北京科学出版社，2000：68
83．荣晓花，凌沛学。溶菌酶的研究进展，中国生化药物杂志，1999，20 (6)：319—320
84．舒馨，陈令新等。毛细管等电聚焦和电渗泵驱动聚焦色谱分离蛋白质，分析试验室，2004。23 (5)：1—4
85．宋宏新，薛海燕，杨芳。鸡蛋壳溶菌酶分离纯化工艺研究，食品工业科技，2003，9：60—63
86．孙怀昌，张泉，施伟庆，李国才，于峰。人溶菌酶cDNA的克隆及其在小鼠体内的表达，中国兽医学报，2004年3月，157—159
87．覃倩萍等。溶菌酶3种测定方法的比较，广西预防医学，1998，4(3)：138
88．覃倩萍，彭崇基等。琼脂糖火箭电泳法测定唾液溶菌酶活性，中国公共卫生，1999， 15 (11)：1040—1041
89．钱世均等。人溶菌酶基冈的合成和克隆，生物工程学报，1994，10(1)：34—38
90．王敏。蛋清提取溶菌酶技术，企业技术开发，2001，8：37—38
91．王跃军，孙谧等。海洋低温溶菌酶的制备及酶学性质，海洋水产研究，2000，21 (4)：54—64
92．卫引茂，苏晓冬等。连续棒状弱阳离子交换柱的合成及其对蛋白质的保留行为，分析化学，2000，28(10)：1194—1198
93．吴晓英，林影，陈慧英。溶菌酶的研究进展，工业微生物，2002年12月，第32卷第4期，55—58
94．吴晓宗，王岁楼，郝莉花。壳聚糖酶的分类及其功能应用现状，食品工业科技，2005，26 (8)：189—192
95．许红，徐奇友，刁新平。绿色添加剂溶菌酶及其应用，饲料添加剂，2005年第26期第2卷，10—13
96．杨鸿藻，刘建国，杨军。溶菌酶两种测定方法的探讨，中华医学检验杂志，1994，17(3)：186
97．杨丽，姜亦文。甲壳质及壳聚糖的微生物降解的现状，青岛化工学院学报，2000，12(4)：301～304
98．叶丹，连宾。溶菌酶及其应用，贵州科学，2003年9月，第21卷第三期，67—70
99．俞伟辉，陈新风，郭荣富，何华西。溶菌酶及其在动物生产中的应用前景，江西饲料，2007年第一期：1—4
100．岳克，新田胜利。马奶溶菌酶的分离和提纯，化学世界，1997，12：666—668
101．张树政，胡学智。酶制剂工业：下册，北京：科学出版社，1984：804～806
102．张伟。滚筒式填料筛板萃取器反胶团法萃取蛋白质，化工学报，2000，12(增刊)：227—231
103．张勇，温其标，胡飞。溶菌酶及其食品工业中的应用，粮油加工与食品机械，2004，3：64—65
104．张宗岩等。溶菌酶——婴儿食品添加剂，中国乳品工业，1993，2l(3)：130-132
105．赵雪雁，佟晓冬，孙颜。SgeamLane SP膨胀床纯化溶菌酶，过程工程学报，2004，4(2)：104—110
106．郅文波，顾铭等。高速逆流色谱技术在生物大分子分离纯化中的应用，生物技术通讯， 2005，16 (1)：96—100
107．邹艳丽，孙谧，王跃军。海洋微生物溶菌酶的纯化与性质研究，生物工程学报，2005，21 (3)：420—424
108．中华人民共和国卫生部药典委员会编。中华人民共和国卫生部药品标准，生化药品，第一册，北京：北京科学出版社，1989：103
109．中华人民共和国卫生部药典委员会编。抗生素生化药品注释第一册，北京：北京科学出版社，1989：10
110．朱奇，陈彦。溶菌酶及其应用，生物学通报，1998，33(10)：9—10
2. Bodmeier R, Chert HG, Paeratakul O.A. Novel approach to the delivery of micro or nanoparticles. Pharm Res, 1989,6 (5): 413-417
3. Calvo P, Remunan-Lopez C, Vila-Jato JL, et al. Novel hydrophilic chitosan/polyethylene oxide nanoparticles as proteins carders. Int J Pharm,2001, 224 (1/2): 159-168
4. Chellat F, Tabrizian M, Dumitriu S, et al. In vitro and in vivo biocompatibility of chitosan-xanthan polyionic complex. J Biomed Res, 2000, 51 (1): 107-116
5. Chen J. et al. Enhancement of the solubility of proteins overexpressed in Escherichia coli by heat shock. J. Mol. Microbiol Biotechnol. 2002, 4 (6):519-524
6. David M. Irwin, Allan C. Wilson. Multiple cDNA Sequences and the Evolution of Bovine Stomach Lysozyme. The Journal of biological chemistry, 1989, 264 (19): 11387～11393
7. Derman AI et al. Mutation that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science, 1993,262 (5140): 1744-1747
8. Dinsmore R.E, et al. OxytetracycLane residues in milk in intrauterine infusion of dairy cows. Boy. Proc, 1994, 26:187-289
9. Hain Ruml udiger, Stenzel Klaus. Use of lysozyme gene structures in plants to increase resistance. Biotechmology Advances, 1997, 15 (3-4): 665
10. Hartley DL, et al. Biochemical Society transactions. 1988,16:101
11. Hayakawa T, ToibanaA, Marumoto R etal. Gene, 1987, 66:53-59
12. Hernandez VP. et al. Characterization and cDNA cloning of an immune-induced lysozyme from cultured Aedes albopictus mosquito cells. Developmental and Comparative Immunology, 2003,27 (1): 11-20
13. Hikima Jun-ichi et al. Molecular cloning, expression and evolution of the Japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein. Biochimica et Biophysica Acta (BBA)/Gene Structure and Expression, 2001,1520 (1): 35-44
14. Hikima S. et al. Characterization and flmction of kuruma shrimp lysozyme possessing lytic activity against Vibrio species. Gene, 2003,316:187-195
15. Hideynki A, Hisham RI, Takeshi K, et al. Bactericidal action of lysozymes attached with various sizes of hydrophobic peptides to the C-terminal using genetic modification. FEBS Letters, 1997,415:114-118
16. Hockney, RC. Recent developments in heterologous protein production in E.coli. Trends Biotech. 1995,13:456-463
17. Horton R, Hunt H, Ho S. Engineering hybrid gene without the use of restriction enzymes: gene splicing by overlap extension. Gene, 1989,77:61-68
18. Kazuhiko Takeuchi, David M.Irwin et al. Multiple cDNA Sequences of Bovine Tracheal Lysozyme. The Journal of biological chemistry, 1993,268 (36): 27440-27446
19. Krueger JK, Stock AM, Schutt CE and Stock J.BioPham. In LM Gierasch and J King eds. Protein foIding:Deciphering the second half of the genetic code.American association for the advancement of science, Washington,D.C, 1990.136-142
20. Lavallie ER, DiBlasio EA, Kovacic S. et al. A thioredoxin gene fusion expression system that circumvents inclusion body formatin in the E.coli cytoplasm. Biotechnology, 1993, 11: 187-193
21. Li B, Huang Y, Paskewitz SM. Hen egg white lysozyme as an inhibitor of mushroom tyrosinase. FEBS Lett. 2006,580 (7): 1877-1882
22. Liu Feng, Wen Zilong. Cloning and expression pattern of the lysozyme C gene in zebrafish. Mechanisms of Development, 2002,113(1): 69-72
23. Luo ZH, Hua ZC. Increased solubility of glutathione S-transferase-P16 (GST-p16) fusion protein by co-expression of chaperones groes and groel in Escherichia coli. Biochem. Mol. Biol. Int. 1998,46 (3): 471-477
24. Maga E, Anderson G, Huang M, et al. Expression of human lysozyme mRNA in the mammary gland oftransgenic mice. Transgenic Research, 1994, 3:36-42
25. Mao HQ, Roy K, Troung Le VL, et al. Chitsan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. J Control Release, 2001,70 (3): 399-421
26. Mafia J, Walter S, Gunther R. Cloning of human lysozyme gene and expression in the saccha romyces cerevisiae.Gene, 1988, 66:223-234
27. Maston AO. The purification of eukaryotic polypeptides synthesized in E.coli. Biochem. J, 1986, 240:1-12
28. Moss JM. et aL Purification, Characterization, and Biosynthesis of Bovine Cartilage Lysozyme Isoforms. Archives of Biochemistry and Biophysics, 1997, 339 (1): 172-182
29. Muraki M, Jigami Y, Tanaka H etal. Agric. Biol. Chem. 1986, 60 (3): 713-723
30. Nakimbugwe D. et al. Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts. FEMS Microbiol. Lett, 2006, 259 (1): 41-46
31. Qan SJ, Yu Y, Tiang KQ. Synthesis and cloning of the human lysozyme gene. Sheng wu Gong cheng Xue bao (Chin. J. Bio. Technol.), 1994, 10(1): 34-38
32．Rebhun WC．等著，赵德明等译。《奶牛疾病学》。北京：中国农业大学出版社，1999，427—432
33. Rinas U. et al. Cysteine to serine substitutions in basic fibmblast growth factor effect on inclusion body formation and proteolytic susceptibility during in vitro refolding. Biotechnology(NY), 1992, 10 (4): 435-440
34. Ruyssen R. Pharmaceutical Enzymes, Properties and Assay Methods. E Storyscientia PVBA, Gent 1978
35. RuttloffH. Industrielle Enzvme Steinkopff, Darmstadt 1979
36．Sambrook J，Fritsch EF,Maniatis T.分子克隆实验指南，第2版，北京：科学出版社，1995
37. Schein CH, Noteborn HM. Formation of solutable recombinant proteins in E.coli is favored by lower growth temperature. Biotechnology, 1988, 6:291-294
38. Shigeru M, Masato K, Masanorl N, et al. Secretion of active human lysozyme by Acremonium chrysogenum using a Fusarium alkaLane protease promoter system. Journal of Biotechnology, 1995, (42): 1-8
39. Tsuchiya K, Tada S, Gomi K et al. App[. Microbiol. Biotechnol. 1992, 38:109-114
40. Usui M. et al. Effective reduction of antigenicity of hen egg lysozyme by site-specific glycosylation. FEBS Letters, 2004, 557 (1-3): 169-173
41. Wang Dianliang. Research Progress in Recombinant Human Lysozyme. China Biotechnology, 2003, 23 (9): 59-63
42. Wen Yi, Irwin David M. Mosaic Evolution of Ruminant Stomach Lysozyme Genes. Molecular Phylogenetics and Evolution, 1999, 13 (3): 474-482
43. Xie Y, Wetlaufer DB. Control of aggregation in protein refolding: the temperature-leap tactic. Prorein Sci, 1996, 5 (3): 517-523
44. Yumiko Shirano et al. Cultivation of E.coli carryinga rice lipoxygenase L-2cDNA produce a soluble and active enzyme at a high level. FEBS Letters, 1990, 271: 128-130
45. Zhang Chun-yan, Ning Bo, Cao Kai-yuan. Cloning ofhCD154 gene from human activated peripheral blood mononuclear cell and expression of hCD154-GST fusion protein in prokaryote. Chinese Journal Of Pathophysiology, 2000, 16 (8): 673-677
46. Zhang Yi-Zheng. Basic experiments in molecular cloning, 1995,11
47．曹访，唐修君，陈丽，王杏龙。奶牛乳腺炎研究进展，黑龙江动物繁殖，2006年，第14卷，第4期，13-15
48．高焕春，吕晓玲，李文英。鸡蛋清溶菌酶提取工艺及其应用初探，天津轻工业学院学报，1996，(1)：37-40
49．胡士明。对奶牛乳房炎发病机制的新认识及临床处理对策的探讨，中国奶牛，1998： 41-44
50．胡松华，杜爱芳，蔡渭明。奶牛临床型和隐性型乳房炎的细菌学分析，中国畜禽传染病，1998，20(4)：199-201
51．贾向志，李元，马文煜。溶菌酶的研究进展，生物技术通讯，2002，13(5)，374-377
52．矫庆华，钱世均，叶军等。人工合成人溶菌酶基因在大肠杆菌中表达，生物工程学报，1997，13(1)：102—104
53．林锋强，潘杭君，胡松华。奶牛乳房炎疫苗研究进展，中国奶牛，2002，1：40—42
54．林向阳，何承云，阮榕生，刘玉环，郑丹丹，朱榕璧。溶菌酶及其应用研究，中国食 品添加剂，2005，No6，105-106
55．李明，孙彦伟。奶牛乳腺炎的防治与研究动态，广东畜牧兽医科技，2006，3，11-14
56．刘宝良，黄俊武。乳房炎的发病原因与自身防御能力，畜牧兽医科技信息，2005，4：67
57．刘玉清，唐兆新。奶牛乳腺炎防治的研究现状，现代畜牧兽医，2006，1，48-50
58．吕俊。溶菌酶治疗子宫内膜炎的试验情况。上海奶牛，2000，24(2)：42—43
59．母安雄，胡松华。奶牛乳房炎抗生素防治失败原因探讨，中国兽医杂志，2002，2：18—20
60．荣晓花，凌沛学。溶菌酶的研究进展，中国生化药物杂志，1999，20(6)：319—320
61．孙怀昌。人溶菌酶基因治疗奶牛乳腺炎的初步研究，畜牧兽医学报，2004，35(2)：227—232
62．孙怀昌。防治奶牛乳房炎的基因工程新药，河北畜牧兽医，2005年第21卷第10期，12-13
63．王慎行。奶牛隐性乳房炎调查及纯中药“消炎增乳灵”治疗试验，中兽医医药杂志，2006，2：38—40
64．谢磊。大肠杆菌表达系统及其研究进展，华南热带农业大学学报，2004，10(2)：16—20
65．杨龙骐，沈永恕，彭义。奶牛乳房炎防治研究进展，郑州牧业工程高等专科学校学报，2001，21(2)：178—181
66．周宇荀，魏东芝。融合蛋白表达载体pGEX及其应用，生命科学，1998，10(3)：122—124
1. Adriana I, Licz A, Laura U, et al. Determination of the fractal dimension of the lysozyme backbone of three diferent organisms, Chaos, Solitons and Fractals, 2001, (12): 757—760
2. Audy P, Quere DL, Leclerc D, et al. Electrophoretic forms of lysozyme activity in various plant species. Phytochemistry, 1990, 29 (4) :1143～1159
3. Bae Sangki, Kim Yonggyun. Lysozyme of the beet arrnyworm, Spodoptera exigua: activity induction and cDNA structure. Comparative Biochemistry and Physiology, Part B, 2003, 135(3): 511-519
4. Bomimcomtro A., Onori G. Investigation by dielectric spectroscopy of domain motions in lysozyme: effect of solvent and binding of inhibitors, Chemical Physics Letters, 2004, 398(1-3): 260-263
5. Caballero M, Ruiz R, Marquez M, et al. Development of a Micmparticle-Enhanced Nephelometric Immunoassay for Quantitation of Human Lysozyme in Pleural Efusion and Plasma, Journal of Clinical Laboratory Analysis, 1999, (13): 301-307
6. Castanon M J, Spevak W, A do lf G R, et al. Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae [J], Gene, 1988,66: 223-234.
7. Chipman DM, Sharon N. Mechanism of lysozyme action. Science, 1969,165:454
8. Conte A, Buonocore GG, Bevilacqua A, Sinigaglia M, Del Nobile MA. Immobilization of lysozyme on polyvinylalcohol films for active packaging applications. J, Food Prot, 2006,69 (4): 866-870
9. Chung L P, Keshav S, Go rdon S, et al. Cloning the human lysozyme cDNA: inverted A lurepeat in the mRNA and in situ hybridization for macrophages and Paneth cell [J], P rocN atlAcad Sci USA, 1988, 85 (17): 6 227-6 231.
10. Diels Ann MJ, et al. Sensitisation of Escherichia coli to antibacterial peptides and enzymes by high-pressure homogenization, International Journal of Food Microbiology, 2005,105 (2): 165-175
11. Durance T D, Nakai S. Simultaneous isolation of avidin and lysozyme from egg albumen [J], Journal of food science, 1988,53 (4): 1096
12. Ghosh R, Cui ZF. Protein purification by ultrafiltration with pretreated membrace [J],Journal of membrane science, 2000,167:47
13. Glazer AN, Barel AO, Howard JB, et al. Isolation and characterization of fig lysozyme. J., Biol. Chem., 1969,244 (13): 3583-3589
14. Hain Ruml udiger, Stenzel Klaus. Use of lysozyme gene structures in plants to increase resistance, Biotechmology Advances, 1997,15 (3-4): 665
15. He LZ, Sun Y. Purification of lysozyme by multistage affinity filtration [J], Bioprocess BiosystEng, 2002,25:155
16. Hernandez VP, et al. Characterization and cDNA cloning of an immune-induced lysozyme from cultured Aedes albopictus mosquito cells, Developmental and Comparative Immunology, 2003,27 (1): 11-20
17. Hikima Jun-ichi et al. Molecular cloning, expression and evolution of the Japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein, Biochimica et BiophysicaActa (BBA)/Gene Structure and Expression, 2001,1520 (1): 35-44
18. Hikima S. et al. Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species, Gene, 2003,316:187-195
19. Howard JB, Giazer AN. Studies of the physicochemical and enzymatic properties of papaya lysozyme. J, Biol. Chem., 1967,242 (24): 5751-5723
20. Huang B, Zhao C, Lei X, et al. The cloning, sequencing and analysis of Chinese human lysozyme gene. cDNA amplified with RT-PCR from human placental total RNA [J ], Chin BiochemJ, 1993,9:2692273.
21. Ibrahim HR. et al. Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function, FEBS Letters. 2001,506 (1): 27-32
22. Ivaanov AE, Galaev IY, Kazakov SV, et al. Thermosensitive copolymers of N-Vinylimidazole as displacers of proteins in immobilized metal aaffinity chromatography [J], Journal of chromatohraphy A, 2001, 907:115
23. Jolles J, Jolles P. Human milk lysozyme: unpublished data concerning the establishment of the complete primary structure; comparison with lysozymes of various o rigins [J], Helv Chim Acta, 1971, 54 (8): 2 668-2 675.
24. Jonathan M. Moss, et.al. Purification, Characterization, and Biosynthesis of Bovine Cartilage Lysozyme Isoforms, Archives of Biochemistry and Biophysics, 1997,339 (1): 172-182
25. Kang HK, Chung YJ, Park CU, Jang YS, Kim BS. Induction of autoimmunity by immunization with hapten-modified hen egg lysozyme in hen egg lysozyme-transgenic mice., Immunology, 2006, 117 (3): 368-378
26. Leheninger AL, Nelson DL, Cox MM. Principles of Biochemistry, 2nd Edition. New York: Worth Publishers, 1993: 180,312
27. Li B, Huang Y, Paskewitz SM. Hen egg white lysozyme as an inhibitor of mushroom tyrosinase, FEBS Lett, 2006, 580 (7): 1877-1882
28. Li chang E, Nakai S, Sim J, et al. Lysozyme sepratation from egg white by cation exchang column chromatography[J], Journal of food science, 1986,51 (4):1 032
29. Liu Feng, Wen Zilong. Cloning and expression pattern of the lysozyme C gene in zebrafish, Mechanisms of Development, 2002,113(1): 69-72
30. Lockey TD, Ourth DD. Purification and Characterization of Lysozyme from Hemolymph of Heliothis virescens Larvae, Biochemical and Biophysical Research Communications, 1996,220 (3): 502-508
31. Lopez-Jaramillo FJ, Perez-Banderas F, Hernandez-Mateo F, Santoyo-Gonzalez F, Production, crystallization and X-ray characterization of chemically glycosylated hen egg-white lysozyme, Acta Crystallograph Sect F Struct Biol Cryst Commun. 2005,61 (Pt 4): 435-438
32. Lord MS. et al. Lysozyme interaction with poly (HEMA)-based hydrogel, Biomaterials, 2006,27(8): 1341-1345
33. Lynn KR. Chitinases and chitobioased form the American lobster. Homarus. Biochem. Physiol., 1990, (96B): 761-766
34. Marcano JG, Tsotisis TT. Catalytic membranes and membrane reactors, Wiley-VCH Verlag GmbH, Weinheim Press, 2002
35. Marc Henke, Gerd Hobom, et.al. Strucral deviations in a bovine low expression lysozyme-ecoding gene active in tissues other than stomach, Gene, 1996,178:131-137
36. Moss JM. et al. Purification, Characterization, and Biosynthesis of Bovine Cartilage Lysozyme Isoforms, Archives of Biochemistry and Biophysics, 1997,339 (1): 172-182
37. Mudgil P, Torres M, Millar TJ. Adsorption of lysozyme to phospholipid and meibomian lipid monolayer films, Colloids Surf B Biointerfaces, 2006,48(2): 128-137
38. Murakand M, et al. Expression of synthetic human lysozyme gene in Escherichia coli, Agricultural Biological Chemistry, 1986,50: 713-723
39. Nattress FM. et al. Evaluation of the ability of lysozyme and nisin to control meat spoilage bacteria, International Jourmal of Food Microbiology, 2001,70 (1-2): 111-119
40. Ohkuri T. et al. Effect of the Structure of the Denatured State of Lysozyme on the Aggregation Reaction at the Early Stages of Folding from the Reduced Form, Journal of Molecular Biology, 2005,347 (1): 159-168
41. Perry LJ, et al. Disulfide bond engineered into T4 lysozyme: stabilization of the protein toward thermal inactivation, Science, 1984,226:555-557
42. Sasagawa N, Saito K, Sugita K. Ionic cross-linking of SO_3H-groupcontaing graft chains helps to capture lysozyme in a permeation mode [J], Journal of chromatography, 1999,848 (1):161
43. Serafica GC, Pimbley J, Belfon G. Protein fraction using fast flow immobilized metal chelate affinity membranes [J], Biotech and Bioeng, 1994,43:21
44. Shireru M, et al. Secretion of active human lysozyme by Acremonium chrysogenum using fusarium alkaLane protease system, Journal of Biotechnology, 1995,45:1-8
45. Sugahara T. et al. Heat denaturation enhanced immunoglobulin production stimulating activity of lysozyme from hen egg white, Biochimica et Biophysica Acta (BBA)/General Subjects, 2002, 1572(1): 19-24
46. Sun HC, Xue FM, Qian K, Fang HX, Qiu HL, Zhang XY, Yin ZH. Intramammary expression and therapeutic effect of a human lysozyme-expressing vector for treating bovine mastitis. J, Zhejiang Univ Sci B, 2006, 7 (4): 324-330
47. Tata SJ, Bemtema JJ, Balabaskaram S. The lysozyme of Hevea brasiliensis latex: isolation, purification, enzymekinetics and a partial amino-acid sequence. J, Rubber Res. Malaya, 1983,31(1): 35-48
48. Usui M. et al. Effective reduction of antigenicity of hen egg lysozyme by site-specific glycosylation, FEBS Letters, 2004,557 (1-3): 169-173
49. Vaidya AA, Lele BS, Kulkarmi MG, et al. Thermmoprecipition of lysozyme from egg white using copolymers of N-isopropylaacrylaamide and acidic monomers[J], Journal of biotechnology, 2001,87:98
50. Vaidya AA, Lele BS, Deshmukh MV, et al. Design and evaluation of new ligands for lysozyme recovery by affinity thermoprecipitation[J], Chemical Engineering Science 2001, 56:5681
51. Vieira MN, Figueroa-Villar JD, Meirelles MN, Ferreira SR, De Felice FG Small Molecule Inhibitors of Lysozyme Amyloid Aggregation, Cell Biochem. Biophys, 2006, 44 (3): 549-553
52. Wen Yi, Irwin David M. Mosaic Evolution of Ruminant Stomach Lysozyme Genes, Molecular Phylogenetics and Evolution, 1999, 13 (3): 474-482
53. Yakobi M, et al. Purifieation and characterization of chiltinase and chitobiase produced by Acromonas hydrophila sup sp, Anaerogenes A52. J. Gen. Appl, Microbiol, Tokyo, 1986, (32): 2538
54. Yoshimura K, Toibana A, N akahama K. Human lysozyme: sequencing of a cDNA, and expression and secretion by Saccharomyces cerevisiae [J], Biochem Biophys Res Commun, 1988, 150(2): 794-801.
55. Youtao Song, et al. In vivo glycosylation suppresses the aggration ofamyloidogenic hen egg white lysozymes esoressed in yeast, FEBS Letters, 2001, 491:63-66
56. Yu Mei, Irwin DM. Evolution of Stomach Lysozyme: The Pig Lysozyme Gene, Molecular Ohylogenetics and Evolution, 1996, 5 (2): 298-308
57. Zhang G, Darius S, Smith SR, Ritchie SJ. In vitro inhibitory effect of hen egg white lysozyme on Clostridium perfringens type A associated with broiler necrotic enteritis and its alpha-toxin production, Lett Appl Microbiol, 2006, 42(2): 138-143
58. Zhang Xing, Studier FW. Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme., Journal of Molecular Biology, 1997, 269 (1): 10-27
59．苍金荣等。微量快速比浊法检测溶菌酶含量，陕西医学检验，1994，9(1)：38—39
60．柴红，陈欢林，刘茉娥。固定金属离子亲和膜分离技术(Ⅱ)研究进展和应用前景[J]，膜科学与技术，1999，19(5)：13
61．陈慧英，吴晓英，林影。溶菌酶分离纯化方法的研究新进展，广东药学院学报，2003，19(4)：355—258
62．船津胜，鹤大典。溶菌酶，济南：山东科技出版社，1982：39～78，257～262
63．戴清源，陈祥贵，李晓霞，张庆。溶菌酶的研究进展，内蒙古农业科技2005(3)：14～16
64．冯惠勇，徐亲民，孙国志。溶菌酶的生物测定方法研究，食品与发酵工业，2002，28(4)：60-64
65．凤权，汤斌。溶菌酶分离纯化方法的研究进展，生物学杂志，2006年2月第23卷第一期，5—7
66．高焕春，吕晓玲，李文英。鸡蛋清溶菌酶提取工艺及其应用初探，天津轻工业学院学报，1996，(1)：37—40
67．高向阳，袁四清。萝卜溶菌酶抑菌效果的初步研究，华南农业大学学报，1997，2：72—75
68．郭新竹。天然肽类防腐剂研究进展，食品与发酵工业，2000，27(2)：72—75
69．郭勇。酶的生产与应用，北京：化学工业出版社，2003，98—101
70．贾向志，袁汉英，马文煜，李元，李育阳。人溶菌酶基因的克隆及其在毕赤酵母中的表达，第四军医大学学报2001；22(22)，2068—2072
71．姜秀云，吴惠毅。平板法测定血清溶菌酶活性影响因素探讨上海医学检验杂志，1994，9(1)：12-13
72．矫庆华等。人工合成人溶菌酶基因在大肠杆菌中表达，生物工程学报，1997，13(1)：102-104
73．柯从玉，白泉，耿信笃。用于变性蛋白质复性及同时纯化的简易型色谱柱的研制及其应用，色谱，2004，22 (4)：394—398
74．李春联。微量溶菌酶比色测定法在临床诊断中的应用，同济大学学报(医学版)，2002，6 (3)：251
75．李国才，孙怀昌，钱科，居杏珠，陈瑾，张泉，朱秋菊。重组人溶菌酶理化及生物学特性的鉴定，中国乳品工业，2005年，Vol 33，No 4，11—14
76．李静，陈欢林，柴红。金属螯合亲和膜吸附分离与纯化溶菌酶的研究，膜科学与技术，2001，21 (4)：5—9
77．林翠花，肖素荣，孟庆国。溶菌酶结构特点及其应用，潍坊学院学报，2005，5 (2)：108-110
78．刘梅，张士璀。溶菌酶C及其生物学功能，生命的化学，2006年26卷5期，465—467
79．刘世清。牛乳中溶菌酶的生物学特性及其应用，畜牧兽医科技信息，2005，4：66—67
80．刘莹，孙荣丹，杨翔，华王丽。溶菌酶的应用现状，饲料工业，2006年第27卷第6期，19—20
81．楼善贤，苗德林。溶菌酶的研究进展，中国肿瘤临床，1994，21(9)：709—711
82．马绪荣，苏德模主编。药品微生物学检验手册，北京：北京科学出版社，2000：68
83．荣晓花，凌沛学。溶菌酶的研究进展，中国生化药物杂志，1999，20 (6)：319—320
84．舒馨，陈令新等。毛细管等电聚焦和电渗泵驱动聚焦色谱分离蛋白质，分析试验室，2004。23 (5)：1—4
85．宋宏新，薛海燕，杨芳。鸡蛋壳溶菌酶分离纯化工艺研究，食品工业科技，2003，9：60—63
86．孙怀昌，张泉，施伟庆，李国才，于峰。人溶菌酶cDNA的克隆及其在小鼠体内的表达，中国兽医学报，2004年3月，157—159
87．覃倩萍等。溶菌酶3种测定方法的比较，广西预防医学，1998，4(3)：138
88．覃倩萍，彭崇基等。琼脂糖火箭电泳法测定唾液溶菌酶活性，中国公共卫生，1999， 15 (11)：1040—1041
89．钱世均等。人溶菌酶基冈的合成和克隆，生物工程学报，1994，10(1)：34—38
90．王敏。蛋清提取溶菌酶技术，企业技术开发，2001，8：37—38
91．王跃军，孙谧等。海洋低温溶菌酶的制备及酶学性质，海洋水产研究，2000，21 (4)：54—64
92．卫引茂，苏晓冬等。连续棒状弱阳离子交换柱的合成及其对蛋白质的保留行为，分析化学，2000，28(10)：1194—1198
93．吴晓英，林影，陈慧英。溶菌酶的研究进展，工业微生物，2002年12月，第32卷第4期，55—58
94．吴晓宗，王岁楼，郝莉花。壳聚糖酶的分类及其功能应用现状，食品工业科技，2005，26 (8)：189—192
95．许红，徐奇友，刁新平。绿色添加剂溶菌酶及其应用，饲料添加剂，2005年第26期第2卷，10—13
96．杨鸿藻，刘建国，杨军。溶菌酶两种测定方法的探讨，中华医学检验杂志，1994，17(3)：186
97．杨丽，姜亦文。甲壳质及壳聚糖的微生物降解的现状，青岛化工学院学报，2000，12(4)：301～304
98．叶丹，连宾。溶菌酶及其应用，贵州科学，2003年9月，第21卷第三期，67—70
99．俞伟辉，陈新风，郭荣富，何华西。溶菌酶及其在动物生产中的应用前景，江西饲料，2007年第一期：1—4
100．岳克，新田胜利。马奶溶菌酶的分离和提纯，化学世界，1997，12：666—668
101．张树政，胡学智。酶制剂工业：下册，北京：科学出版社，1984：804～806
102．张伟。滚筒式填料筛板萃取器反胶团法萃取蛋白质，化工学报，2000，12(增刊)：227—231
103．张勇，温其标，胡飞。溶菌酶及其食品工业中的应用，粮油加工与食品机械，2004，3：64—65
104．张宗岩等。溶菌酶——婴儿食品添加剂，中国乳品工业，1993，2l(3)：130-132
105．赵雪雁，佟晓冬，孙颜。SgeamLane SP膨胀床纯化溶菌酶，过程工程学报，2004，4(2)：104—110
106．郅文波，顾铭等。高速逆流色谱技术在生物大分子分离纯化中的应用，生物技术通讯， 2005，16 (1)：96—100
107．邹艳丽，孙谧，王跃军。海洋微生物溶菌酶的纯化与性质研究，生物工程学报，2005，21 (3)：420—424
108．中华人民共和国卫生部药典委员会编。中华人民共和国卫生部药品标准，生化药品，第一册，北京：北京科学出版社，1989：103
109．中华人民共和国卫生部药典委员会编。抗生素生化药品注释第一册，北京：北京科学出版社，1989：10
110．朱奇，陈彦。溶菌酶及其应用，生物学通报，1998，33(10)：9—10