利用生物反应器表达重组人溶菌酶
|
摘要
溶菌酶(lysozyme, LYZ)是自然界普遍存在的一种酶,因其能溶解细菌细胞壁具有溶菌作用而得名。溶菌酶根据分子结构、分子量和来源的不同可分为六类:植物溶菌酶、微生物溶菌酶、噬菌体溶菌酶、c型、g型和I型3种动物溶菌酶,其中c型、g型和噬菌体溶菌酶最常见。溶菌酶是人和动物体液和组织中的一种防御因子,在非特异性免疫中具有重要作用,具有抗菌、消炎、促进组织修复和增强免疫力等多种药理作用,因而广泛应用于食品、饲料工业、科研和医学临床中。
人溶菌酶(human lysozyme,hLYZ)属于c型溶菌酶,由130个氨基酸组成,分子量为14.4kD,含有4个二硫键;与其它溶菌酶相比,其一级结构差异较大,但高级结构非常相似。人溶菌酶与目前临床上应用最多的鸡蛋清溶菌酶相比具有以下优势:人溶菌酶是人体内的蛋白,具有天然的相容性,没有毒性和副作用;人溶菌酶比鸡蛋清溶菌酶的杀菌活性高3倍,而且热稳定性也要远高于鸡蛋清溶菌酶;人溶菌酶还有多种特有的与其自身的催化作用无关的理化作用,如抗病毒和抗肿瘤。随着人们对人溶菌酶功能的深入了解和需求的增加,其大规模的生产和应用越来越受到重视。
目前应用的人溶菌酶大部分是从人乳和胎盘中提取,由于受到来源和规模的限制,存在着人溶菌酶产量不高、质量不稳定且生产成本昂贵等问题。利用生物反应器生产人溶菌酶不仅对上述问题的解决具有积极作用,也为人溶菌酶的生产提供了一条可选择的途径。
本研究尝试利用生物反应器来表达重组人溶菌酶,所用的生物反应器包括大肠杆菌、毕赤酵母和植物油体三种表达系统,取得了如下研究结果:①分别按照大肠杆菌和植物偏爱密码子设计并合成了人溶菌酶基因,为了便于后期分离纯化,设计时在基因中引入了his-tag和肠激酶酶切位点;②成功构建了大肠杆菌表达载体pET32a-hLYZe,并转化入大肠杆菌表达型菌株BL21,获得了含有人溶菌酶基因的工程菌株,并通过IPTG诱导表达出了重组人溶菌酶蛋白,表达产物以包涵体形式存在,经过包涵体变性、溶解、复性后,通过SDS-PAGE和Western blot检测,证明重组蛋白在大肠杆菌中成功表达,且具有免疫原性,经过镍亲和层析柱纯化了重组蛋白,通过比浊法确认了其具有生物学活性;③成功构建了毕赤酵母分泌型表达载体pPICZ?-hLYZ,转化毕赤酵母GS115获得了工程菌株,利用甲醇诱导表达了重组人溶菌酶蛋白,通过SDS-PAGE和Western blot检测,证明重组蛋白得到表达,且具有免疫原性,重组蛋白经过镍亲和层析柱纯化后,得到了初步纯化的人溶菌酶蛋白,用Bradford法测得其含量为324mg/L,通过酶标仪快速比浊法测得毕赤酵母表达的重组人溶菌酶蛋白活性为4473U/mL,比活性约为13800U/mg,高于通过人乳提取等常规方法获得的人溶菌酶蛋白;④成功构建了含“twin T-DNAs”的植物双元表达载体OP::(his)-OG-hLYZ,转化入根癌农杆菌( Agrobacterium tumefaciens)GV3101中获得了基因工程菌,通过农杆菌介导法转化拟南芥,共获得了27株卡那霉素抗性植株。
Lysozyme (LYZ) is a ubiquitous enzyme in the nature. It can kill bacterium by lysing the bacterial cell wall. Lysozymes are classified into six types according to their molecular structures, molecular weights and sources: plant type lysozyme, bacteria type lysozyme, phage lysozyme, chicken egg-white lysozyme (c-type, clyz), goose egg-white lysozyme (g-tpye, glyz) and invertebrate type lysozyme (i-type). The most common ones in the nature are c-type, g-type and phage lysozymes. Lysozyme is an important defensive factor presented in the humor body liquid and tissues of human and other animals and plays a significant role in the non-specific immunity. It also has many other pharmacological functions, such as anti-bacterium, anti-inflammation, promoting organism recovery, so it is widely used in food and feed industries, scientific research and medical clinical treatments.
Human lysozyme (hLYZ) is a 130-animo acid c-type lysozyme with 4 disulfide bonds, and its molecular weight is 14.4kD. Comparing with other kinds of lysozyme, hLYZ has a much different primary structure, but has a very similar tertiary structure. Comparing with chicken egg-white lysozyme which is widely used in clinic, hLYZ has many advantages: it is a natural protein in human body, so it is safe and harmless when used as a drug; its bioactivity to kill bacterium is three times higher than chicken egg-white lysozyme, and its thermal stability is much higher, too; it also has many other specific important functions which are not related with its catalysis, such as anti-virus and anti-tumor and improving immunization. With the in-depth understanding of hLYZ functions and the increasing demand in the market, more and more attention is paid in the large-scale production and application of hLYZ.
Currently, most hLYZ is extracted from human milk and placenta. Limited by the source and scale, the problems for production of hLYZ are low yield, unstable quality and high cost. Utilizing bioreactor to produce hLYZ can make positive contribution to solve these problems, and can also provide an alternative way to hLYZ.
The present study tried to use bioreactors to express recombinant hLYZ in three different expression systems: Escherichia coli, Pichia pastoris and plant oil body. The major achievements in this study were as follows:①two different hLYZ genes were designed and synthesized according to the plant and E.coli codon bias usages, respectively, and in both genes a his-tag and a enterokinase cleavage site were introduced to simplify the purification procedures later on.②an E. coli expression vector pET32a-hLYZe was successfully constructed, which was subsequently transferred into E. coli strain BL21. After induction by IPTG, the engineered strain expressed recombinant hLYZ with specific immunogenicity, which existed as insoluble inclusion bodies, which was confirmed by SDS-PAGE and Western blot analyses. The recombinant protein was purified by Ni+ affinity chromatography after a series of denaturation, solution and renaturation procedures, and then analyzed for the hLYZ bioactivity by turbidimetric method. The result showed that the recombinant hLYZ expressed by E. coli was bioactive.③a P. pastoris secreton expression vector pPICZ?-hLYZ was successfully constructed, which was then transferred into P. pastoris strain GS115. After induction by methanol, the engineered strain expressed recombinant hLYZ with specific immunogenicity, which was confirmed by SDS-PAGE and Western blot analyses. The recombinant protein was purified by Ni+ affinity chromatography and measured for hLYZ concentration by Bradford assay, and then analyzed for the hLYZ bioactivity by turbidimetric method. The result indicated that the concentration of expressed hLYZ was 324mg/L, the bioactivity was 4473U/mL, and the specific activity was approximately 13800U/mg, which was higher than conventional hLYZ extracted from human milk.④a“Twin T-DNAs”-containing plant expression vector p2300-OP-(his)-OG-hLYZ was successfully constructed, which was then transferred into Agrobacterium tumefaciens strain GV3101. Twenty-seven kanamycin-resistant Arabidopsis plant were regenerated by Agrobacterium-mediated transformation.
人溶菌酶(human lysozyme,hLYZ)属于c型溶菌酶,由130个氨基酸组成,分子量为14.4kD,含有4个二硫键;与其它溶菌酶相比,其一级结构差异较大,但高级结构非常相似。人溶菌酶与目前临床上应用最多的鸡蛋清溶菌酶相比具有以下优势:人溶菌酶是人体内的蛋白,具有天然的相容性,没有毒性和副作用;人溶菌酶比鸡蛋清溶菌酶的杀菌活性高3倍,而且热稳定性也要远高于鸡蛋清溶菌酶;人溶菌酶还有多种特有的与其自身的催化作用无关的理化作用,如抗病毒和抗肿瘤。随着人们对人溶菌酶功能的深入了解和需求的增加,其大规模的生产和应用越来越受到重视。
目前应用的人溶菌酶大部分是从人乳和胎盘中提取,由于受到来源和规模的限制,存在着人溶菌酶产量不高、质量不稳定且生产成本昂贵等问题。利用生物反应器生产人溶菌酶不仅对上述问题的解决具有积极作用,也为人溶菌酶的生产提供了一条可选择的途径。
本研究尝试利用生物反应器来表达重组人溶菌酶,所用的生物反应器包括大肠杆菌、毕赤酵母和植物油体三种表达系统,取得了如下研究结果:①分别按照大肠杆菌和植物偏爱密码子设计并合成了人溶菌酶基因,为了便于后期分离纯化,设计时在基因中引入了his-tag和肠激酶酶切位点;②成功构建了大肠杆菌表达载体pET32a-hLYZe,并转化入大肠杆菌表达型菌株BL21,获得了含有人溶菌酶基因的工程菌株,并通过IPTG诱导表达出了重组人溶菌酶蛋白,表达产物以包涵体形式存在,经过包涵体变性、溶解、复性后,通过SDS-PAGE和Western blot检测,证明重组蛋白在大肠杆菌中成功表达,且具有免疫原性,经过镍亲和层析柱纯化了重组蛋白,通过比浊法确认了其具有生物学活性;③成功构建了毕赤酵母分泌型表达载体pPICZ?-hLYZ,转化毕赤酵母GS115获得了工程菌株,利用甲醇诱导表达了重组人溶菌酶蛋白,通过SDS-PAGE和Western blot检测,证明重组蛋白得到表达,且具有免疫原性,重组蛋白经过镍亲和层析柱纯化后,得到了初步纯化的人溶菌酶蛋白,用Bradford法测得其含量为324mg/L,通过酶标仪快速比浊法测得毕赤酵母表达的重组人溶菌酶蛋白活性为4473U/mL,比活性约为13800U/mg,高于通过人乳提取等常规方法获得的人溶菌酶蛋白;④成功构建了含“twin T-DNAs”的植物双元表达载体OP::(his)-OG-hLYZ,转化入根癌农杆菌( Agrobacterium tumefaciens)GV3101中获得了基因工程菌,通过农杆菌介导法转化拟南芥,共获得了27株卡那霉素抗性植株。
Lysozyme (LYZ) is a ubiquitous enzyme in the nature. It can kill bacterium by lysing the bacterial cell wall. Lysozymes are classified into six types according to their molecular structures, molecular weights and sources: plant type lysozyme, bacteria type lysozyme, phage lysozyme, chicken egg-white lysozyme (c-type, clyz), goose egg-white lysozyme (g-tpye, glyz) and invertebrate type lysozyme (i-type). The most common ones in the nature are c-type, g-type and phage lysozymes. Lysozyme is an important defensive factor presented in the humor body liquid and tissues of human and other animals and plays a significant role in the non-specific immunity. It also has many other pharmacological functions, such as anti-bacterium, anti-inflammation, promoting organism recovery, so it is widely used in food and feed industries, scientific research and medical clinical treatments.
Human lysozyme (hLYZ) is a 130-animo acid c-type lysozyme with 4 disulfide bonds, and its molecular weight is 14.4kD. Comparing with other kinds of lysozyme, hLYZ has a much different primary structure, but has a very similar tertiary structure. Comparing with chicken egg-white lysozyme which is widely used in clinic, hLYZ has many advantages: it is a natural protein in human body, so it is safe and harmless when used as a drug; its bioactivity to kill bacterium is three times higher than chicken egg-white lysozyme, and its thermal stability is much higher, too; it also has many other specific important functions which are not related with its catalysis, such as anti-virus and anti-tumor and improving immunization. With the in-depth understanding of hLYZ functions and the increasing demand in the market, more and more attention is paid in the large-scale production and application of hLYZ.
Currently, most hLYZ is extracted from human milk and placenta. Limited by the source and scale, the problems for production of hLYZ are low yield, unstable quality and high cost. Utilizing bioreactor to produce hLYZ can make positive contribution to solve these problems, and can also provide an alternative way to hLYZ.
The present study tried to use bioreactors to express recombinant hLYZ in three different expression systems: Escherichia coli, Pichia pastoris and plant oil body. The major achievements in this study were as follows:①two different hLYZ genes were designed and synthesized according to the plant and E.coli codon bias usages, respectively, and in both genes a his-tag and a enterokinase cleavage site were introduced to simplify the purification procedures later on.②an E. coli expression vector pET32a-hLYZe was successfully constructed, which was subsequently transferred into E. coli strain BL21. After induction by IPTG, the engineered strain expressed recombinant hLYZ with specific immunogenicity, which existed as insoluble inclusion bodies, which was confirmed by SDS-PAGE and Western blot analyses. The recombinant protein was purified by Ni+ affinity chromatography after a series of denaturation, solution and renaturation procedures, and then analyzed for the hLYZ bioactivity by turbidimetric method. The result showed that the recombinant hLYZ expressed by E. coli was bioactive.③a P. pastoris secreton expression vector pPICZ?-hLYZ was successfully constructed, which was then transferred into P. pastoris strain GS115. After induction by methanol, the engineered strain expressed recombinant hLYZ with specific immunogenicity, which was confirmed by SDS-PAGE and Western blot analyses. The recombinant protein was purified by Ni+ affinity chromatography and measured for hLYZ concentration by Bradford assay, and then analyzed for the hLYZ bioactivity by turbidimetric method. The result indicated that the concentration of expressed hLYZ was 324mg/L, the bioactivity was 4473U/mL, and the specific activity was approximately 13800U/mg, which was higher than conventional hLYZ extracted from human milk.④a“Twin T-DNAs”-containing plant expression vector p2300-OP-(his)-OG-hLYZ was successfully constructed, which was then transferred into Agrobacterium tumefaciens strain GV3101. Twenty-seven kanamycin-resistant Arabidopsis plant were regenerated by Agrobacterium-mediated transformation.
引文
[1] Pellegrini A, Thomas U, von Fellenberg R, et al. Bactericidal activities of lysozyme and aprotinin against gram~negative and gram~positive bacteria related to their basic character. Journal of Applied Bacteriology.1992,72(3):180~187.
[2] Masuda T, Ide N, Kitabatake N. Effects of chemical modification of lysine residues on the sweetness oflysozyme. Chemical Senses.2005,30(3):253~264.
[3] Fleming A. On a remarkable bacteriolytic element found in tissues and secretions. Proceedings of the Royal Society of London.1922,93(653):306~317.
[4] Lonnerdal B. Biochemistry and physiological function of human milk proteins. Clinical. Nutrition.1985,42:1299~1317.
[5] Weaver H, Grutter G, Remington J. Comparison of goose-type, chicken-type, and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution. Journal of Molecular Evolution.1985,21(2):97~111.
[6] Prager E, Wilson A, Amheim N, et.al. Widespread Distribution of Lysozyme in egg white of birds.Journal of Biochemistry.1974,249(22):7295~7297.
[7] Schoentgen F, Jolles J, Jolles P, et al. Complete amino acid sequence of ostrich egg-white lysozyme,a goose-type lysozyme.European Journal of Biochemistry.1982,123(3):489~497.
[8] Maattheus B, Crutter M, Anderson W, et al. Common precursor of lysozyme of hen egg-white and bacteriophage T4. Nature.1981,290:334~340.
[9] Grutter M. Goose lysozyme structure: an evolution link between hen and bacteriophage lysozyme. Nature.1983,303:828~831.
[10] Muraki M, Goda S, Nagahora H, et al. Importance of Van der Waals contact between Glu 35 and Trp 109 to the catalytic action of human lysozyme. Protein Science.1997,6(2):473~476.
[11] Veenstra D, Kollman P. Modeling protein stability: a theoretical analysis of the lysozyme mutants. Protein Engineering.1997,10(7):789~796.
[12] Enes D, Mackenzie D, Archer D. Transcriptional and post-transcriptional events affect the production of secreted hen egg white lysozyme by Aspergillus niger. Transgenicresearch.1994,3(5):297~303.
[13] Peters C, Kruse U, Pollwein R, et al. The human lysozyme gene: sequence organization and chromosomal localization. European Journal of Biochemistry.1989,182(33):507~516.
[14] Alexander J, Albrecht E, Manuel G, et al. Exon encode functional and structure units of chicken lysozyme. Proceedings of the National Academy of Sciences.1980,77(10):5759~5868.
[15] Baniahmad A, Steiner C, Renkawitz R. Modular structure of a chicken lysozyme silencer: Involvement of an unusual thyroid hormone receptor binding site. Cell.1990,61(4):505~514.
[16] Alexander J, Albrecht E, Manuel G. Exons encode functional and structural units of chicken lysozyme. Proceedings of the National Academy of Sciences.1980,77(10):5759~5763.
[17] Houser M. Improved turbidimetric assay for lysozyme inurine. Clinical Chemistry.1983,29(7):1488~1492.
[18] Ewan W, Ludmilla A, Duncan A, et al. A raman optical activity study of the amyloidogenic prefibrillar intermediate of human lysozyme.Journal of Molecular Biology.2000,301(2):553~563.
[19] Leo I, Steven D, Nell E, et al. Modulation of neuotrophil function of lysozyme. Journal of Clinical Investigation.1979,64:226~232.
[20] Zhao L, Tan X. Effect Mechanism of structure-changed water on heat stability of lysozyme. Transactions of Tianjin University.2003,9(3):177~179.
[21] 许红, 徐奇友, 刁新平. 绿色添加剂溶菌酶及其应用. 饲料工业.2005,26(2):62~64.
[22] 王佃亮. 重组人溶菌酶的研究进展. 中国生物工程杂志.2003,23(9):59~63.
[23] 张凤凯. 溶菌酶及食品保鲜剂的应用. 肉类研究.2001,(4):41~42.
[24] 方元超. 溶菌酶及其应用前景. 中国食品添加剂.1999,(4):39~43.
[25] 邹仕庚, 朱光道. 溶菌酶及其在畜禽饲料中的应用.畜禽业.2003, (2):219~223.
[26] 王欣平. 含溶菌酶牙膏.牙膏工业.1994(1):31~ 34.
[27] 姚志麒. 环境卫生学. 北京: 人民卫生出版社.1988:89~92.
[28] Yu Q, Li J. Lysozyme and its applications. Academic Periodical of Farm Products Processing.2007,(109):34~38.
[29] 叶丹, 连宾. 溶菌酶及其应用.贵州科学.2003,21(3):67~70.
[30] 郝 杰 . 海 洋 溶 菌 酶 酶 学 性 质 及 其 在 化 妆 品 中 的 抑 菌 效 果 . 日 用 化 学 工业.2004,36(4):366~369.
[31] Adriana I, Licz A, Laura U, et al. Determination of the fractal dimension of the lysozymebackbone of three different organisms.Chaos, Solitons and ractals.2001,(12):757~760.
[32] Mark A, Van Gunsteren F. Simulation of the thermal denaturation of hen egg white lysozyme: trapping the molten globule state. Biochemistry(Easton).1992,31(3434):7745~7748.
[33] 叶军, 钱世钧. 工程菌人溶菌酶的纯化和性质.微生物学报.1999 ,39 (1):55～59.
[34] Osserman E, Klockars M, Halper G, et al. Effects of lysozyme on normal and transformed mammalian cells. Nature.1973,243:331~335.
[35] Warren J, Rinehart J, Zwilling B, et al. Lysozyme enhancement of tumor cell immunoprotection in a murine fibrosarcoma. Cancer Research.1981,41:1642~1645.
[36] Nakajima H, Muranaka T, Ishige F, et al. Fungal and bacterial disease resistance in transgenic plants expressing human lysozyme. Plant Cell Reports.1997,16(7): 674~679.
[37] Pei X, Chen S, Jia S, et al. Creation of transgenic bananas expressing human lysozyme gene for panama wilt resistance. Journal of Integrative Plant Biology.2005,47(8):971~977.
[38] Huang J, Nandi S, Huang N, et al. Expression of natural antimicrobial human lysozyme in rice grains. Molecular Breeding.2002,10:83~94.
[39] Takaichi M, Oeda K. Transgenic carrots with enhanced resistance against two major pathogens. Plant Science.2000,153(2):135~144.
[40] Anderson G. Dairy cattle with a gene for human lysozyme. California Diary Research Foundation.1999, Project number: CDRF#ANG96-1.
[41] Maga E, Anderson G, Murray J. The effect of mammary gland expression of human lysozyme on the properities of milk from transgenic mice. Journal of Dairy Science.1995,78:2645 ~2652.
[42] Roberts B, Tullio P, Vitale J, et al.. Cloning of the goat betacasein-encoding gene and expression in transgenic mice. Gene.1992,121:255~262.
[43] Yu Z, Fan B, Li N, et al. Expression of recombinant human lysozyme in the milk of transgenic mice. Chinese Science Bulletin.2003, 48(21):2331~2335.
[44] 李国才, 孙怀昌, 孙强. 乳腺高效表达人溶菌酶转基因小鼠的制备.中华微生物学与免疫学杂志.2004,24(2):142~145.
[45] Murakami M, Jigani Y, Tanaka H, et al. Expression of synthetic human lysozyme gene in Escherichia coli. Agricultural Biological Chemistry.1986,(50):713~723.
[46] Yoshimura K, Toibana A, Nakahama K. Human lysozyme: Sequence of a cDNA, andexpression and secretion by Saccharomyces cerevisae. Biochemistry and Biophysics Research Communication.1988,(105):794～801.
[47] 贾向志, 袁汉英, 马文熠等. 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.
[48] 钱世钧, 陈军, 叶欣等. 从大肠杆菌包含体中提取有活性的人溶菌酶的研究. 生物工程学报.1996 ,12(增刊) :266～268.
[49] Fisher R, Emans N. Molecular farming of pharmaceutical proteins. Transgenic Research.2000,9:279~299.
[50] 焦瑞身. 展望即将到来的“分子农业”.生物工程学报.2001,17(4):361~364.
[51] Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants.Nature.1989,342:76~78.
[52] Ma J, Hein M. Immunotherpeuic potential of antibodies produced in plants. Trends Biotechnology.1995,13:522~527.
[53] Verwoerd T, Paridon P, Ooyen A. Stable accumulation of aspergillus niger phytase in transgenic tobacco leaves. Plant Physiology.1995,109:1199~1205.
[54] Magnuson N, Linzmaier P, Reeves R, et al. Secretion of biologically active human interleukin-2 and interleukin-4 from genetically modified tobacco cells in suspension culture. Protein Exprission and Purifiration.1998,13:45~52.
[55] Sijmons P, Dekker B, Schrammeijer B, et al. Production of correctly processed human serum albumin in transgenic plants. Biotechnology.1990,8:217~221.
[56] Walmsley A, Arntzen C. Plants for delivery of edible vaccines. Current Opinion in Biotechnology.2000,11:126~129.
[57] Fischer R, Stoger E, Schillberg S, et al. Plant-based production of biopharmaceuticals. Current Opinion in Plant Biology.2004,7(2):152~158.
[58] Walsh G. Pharmaceuticals, biologics and biopharmaceuticals. Biopharmaceuticals, Biochemistry and Biotechnology.Wiley, Chichester,UK.1998:1~35.
[59] 邱信芳. 人凝血因子 IX 基因乳腺特异表达载体的构建及其在奶山羊乳腺中的分泌性表达.生物工程学报.1997, 2:78~83.
[60] Echelard Y. Recombinant protein production in transgenic animals. Current Opinion in Biotechnology.1996,7(5):536~540.
[61] Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants. Nature.1989,342:76~78.
[62] Voss A, Niersbach M, Hain R, et al. Reduced virus infectivity in N. tabacum secreting a MV-specific full size antibody. Molecular Breeding.1995,1:39~50.
[63] Cabanes M, Fitchette A, Loutelier B, et al. N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants. Glycobiology.1999, 9:365~372.
[64] Ohya K, Itchoda N, Ohashi K, et al. Expression of biologically active human tumor necrosis factor~alpha in transgenic potato plant. Interferon Cytokine Research.2002,22(3):371~378.
[65] Ziegler M, Thomas S, Danna K. Accumulation of a thermostable endo-1, 4-b-D-glucanase in the apoplast of Arabidopsis thaliana leaves. Molecular Breedding. 2000,6:37~41.
[66] Sambrook J, Fritsch F, Maniatis T. Molecular Cloning: A laboratory manual.3rd ed. New York: Cold Spring Harbor Laboratory.
[67] Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Analysis Biochemistry.1976,72(1~2):248~254.
[68] 苍金荣 , 刘万里 , 刘延龄等 . 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.
[69] 王关林, 方宏筠.植物基因工程.北京:科学出版社.2002,8:397~400.
[70] Lin R, Ding Z, Kuang T, et al. Plant Molecular Biology.2001,19:379a~379e
[71] Andersson S and Kurland C, Codon preferences in free-living microorganisms. Microbiology Review.1990,54(2):198~210.
[72] Mason H, Haq T, Clements J, et al. Edible vaccine protects mice against E.coli heat-labile enterotoxin: potatoes expressing a synthetic LT-B gene.Vaccine.1998,16(13):1336~1343.
[73] Julian K, Drake P. The procuction of recombinant pharmaceutical proteins in plants. Genetics.2003,4:794~805.
[74] Matthews P, Wang M, Waterhouse P, et al. Marker gene elimination from transgenic barley, using co-transformation with adjacent ?twin T-DNAs? on a standard Agrobacterium transformation vector. Molecular Breeding. 2001,7:195~202.
[75] Dale E, Ow D. Gene transfer with subsequent removal of the selection gene from the plant genome. Proceedings of the National Academy of Sciences.1991,88:10558~10562.
[76] Goldsbrough A, Lastrella C, Yoder J. Transposition mediated repositioning and subsequent elimination of marker genes from transgenic tomato.Biotechnology.1993,11:1286~1292.
[77] 王永飞, 马三梅, 亦如瀚. 转基因植物选择标记基因及其安全性问题.植物生理学通讯. 2004,40(5):604~610.
[78] Yoder J, Goldsbrough A. Transfomation systems for gerneration marker-free transgenic plants. Biotechnology.1994,12:263~267.
[79] De Block M, Debrouwer D. Two T-DNAs cotransformed into Brassica napus by a double Agrobacterium tumefaciens infection are mainly integrated at the same locus. Theory Applied Genetics.1991,82:257~263.
[80] Komari T, Hiei Y, Saito Y, et al. Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant Journal.1996,10:165~174.
[81] Lu H, Zhou X, Gong Z, et al. Generation of selectable marker-free transgenic rice using double right borders binary vectors. Plant Physiology.2001,28:241~248.
[82] Miller M, Tagliani L, Wang N, et al. High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system. Transgenic Research.2002,11:381~396.
[83] Frandsen G, Mundy J, Tzen J. Oil bodies and their associated proteins, oleosin and caleosin. Physiologia Plantarum.2001,112 (3):301~307.
[84] 刘 昱 辉 , 贾 士 荣 . 植 物 油 体 表 达 体 系 的 研 究 进 展 . 农 业 生 物 技 术 学报.2003,11(5):531~537.
[85] 曲勍, 李校堃, 于雅琴. 利用油体表达系统生产外源重组蛋白.中国生物工程杂志.2007,27(8):111～115.
[86] 陈琳, 张亚东. 嗜甲醇酵母表达系统研究进展.国外医学.2002,24(1):11~14.
[2] Masuda T, Ide N, Kitabatake N. Effects of chemical modification of lysine residues on the sweetness oflysozyme. Chemical Senses.2005,30(3):253~264.
[3] Fleming A. On a remarkable bacteriolytic element found in tissues and secretions. Proceedings of the Royal Society of London.1922,93(653):306~317.
[4] Lonnerdal B. Biochemistry and physiological function of human milk proteins. Clinical. Nutrition.1985,42:1299~1317.
[5] Weaver H, Grutter G, Remington J. Comparison of goose-type, chicken-type, and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution. Journal of Molecular Evolution.1985,21(2):97~111.
[6] Prager E, Wilson A, Amheim N, et.al. Widespread Distribution of Lysozyme in egg white of birds.Journal of Biochemistry.1974,249(22):7295~7297.
[7] Schoentgen F, Jolles J, Jolles P, et al. Complete amino acid sequence of ostrich egg-white lysozyme,a goose-type lysozyme.European Journal of Biochemistry.1982,123(3):489~497.
[8] Maattheus B, Crutter M, Anderson W, et al. Common precursor of lysozyme of hen egg-white and bacteriophage T4. Nature.1981,290:334~340.
[9] Grutter M. Goose lysozyme structure: an evolution link between hen and bacteriophage lysozyme. Nature.1983,303:828~831.
[10] Muraki M, Goda S, Nagahora H, et al. Importance of Van der Waals contact between Glu 35 and Trp 109 to the catalytic action of human lysozyme. Protein Science.1997,6(2):473~476.
[11] Veenstra D, Kollman P. Modeling protein stability: a theoretical analysis of the lysozyme mutants. Protein Engineering.1997,10(7):789~796.
[12] Enes D, Mackenzie D, Archer D. Transcriptional and post-transcriptional events affect the production of secreted hen egg white lysozyme by Aspergillus niger. Transgenicresearch.1994,3(5):297~303.
[13] Peters C, Kruse U, Pollwein R, et al. The human lysozyme gene: sequence organization and chromosomal localization. European Journal of Biochemistry.1989,182(33):507~516.
[14] Alexander J, Albrecht E, Manuel G, et al. Exon encode functional and structure units of chicken lysozyme. Proceedings of the National Academy of Sciences.1980,77(10):5759~5868.
[15] Baniahmad A, Steiner C, Renkawitz R. Modular structure of a chicken lysozyme silencer: Involvement of an unusual thyroid hormone receptor binding site. Cell.1990,61(4):505~514.
[16] Alexander J, Albrecht E, Manuel G. Exons encode functional and structural units of chicken lysozyme. Proceedings of the National Academy of Sciences.1980,77(10):5759~5763.
[17] Houser M. Improved turbidimetric assay for lysozyme inurine. Clinical Chemistry.1983,29(7):1488~1492.
[18] Ewan W, Ludmilla A, Duncan A, et al. A raman optical activity study of the amyloidogenic prefibrillar intermediate of human lysozyme.Journal of Molecular Biology.2000,301(2):553~563.
[19] Leo I, Steven D, Nell E, et al. Modulation of neuotrophil function of lysozyme. Journal of Clinical Investigation.1979,64:226~232.
[20] Zhao L, Tan X. Effect Mechanism of structure-changed water on heat stability of lysozyme. Transactions of Tianjin University.2003,9(3):177~179.
[21] 许红, 徐奇友, 刁新平. 绿色添加剂溶菌酶及其应用. 饲料工业.2005,26(2):62~64.
[22] 王佃亮. 重组人溶菌酶的研究进展. 中国生物工程杂志.2003,23(9):59~63.
[23] 张凤凯. 溶菌酶及食品保鲜剂的应用. 肉类研究.2001,(4):41~42.
[24] 方元超. 溶菌酶及其应用前景. 中国食品添加剂.1999,(4):39~43.
[25] 邹仕庚, 朱光道. 溶菌酶及其在畜禽饲料中的应用.畜禽业.2003, (2):219~223.
[26] 王欣平. 含溶菌酶牙膏.牙膏工业.1994(1):31~ 34.
[27] 姚志麒. 环境卫生学. 北京: 人民卫生出版社.1988:89~92.
[28] Yu Q, Li J. Lysozyme and its applications. Academic Periodical of Farm Products Processing.2007,(109):34~38.
[29] 叶丹, 连宾. 溶菌酶及其应用.贵州科学.2003,21(3):67~70.
[30] 郝 杰 . 海 洋 溶 菌 酶 酶 学 性 质 及 其 在 化 妆 品 中 的 抑 菌 效 果 . 日 用 化 学 工业.2004,36(4):366~369.
[31] Adriana I, Licz A, Laura U, et al. Determination of the fractal dimension of the lysozymebackbone of three different organisms.Chaos, Solitons and ractals.2001,(12):757~760.
[32] Mark A, Van Gunsteren F. Simulation of the thermal denaturation of hen egg white lysozyme: trapping the molten globule state. Biochemistry(Easton).1992,31(3434):7745~7748.
[33] 叶军, 钱世钧. 工程菌人溶菌酶的纯化和性质.微生物学报.1999 ,39 (1):55～59.
[34] Osserman E, Klockars M, Halper G, et al. Effects of lysozyme on normal and transformed mammalian cells. Nature.1973,243:331~335.
[35] Warren J, Rinehart J, Zwilling B, et al. Lysozyme enhancement of tumor cell immunoprotection in a murine fibrosarcoma. Cancer Research.1981,41:1642~1645.
[36] Nakajima H, Muranaka T, Ishige F, et al. Fungal and bacterial disease resistance in transgenic plants expressing human lysozyme. Plant Cell Reports.1997,16(7): 674~679.
[37] Pei X, Chen S, Jia S, et al. Creation of transgenic bananas expressing human lysozyme gene for panama wilt resistance. Journal of Integrative Plant Biology.2005,47(8):971~977.
[38] Huang J, Nandi S, Huang N, et al. Expression of natural antimicrobial human lysozyme in rice grains. Molecular Breeding.2002,10:83~94.
[39] Takaichi M, Oeda K. Transgenic carrots with enhanced resistance against two major pathogens. Plant Science.2000,153(2):135~144.
[40] Anderson G. Dairy cattle with a gene for human lysozyme. California Diary Research Foundation.1999, Project number: CDRF#ANG96-1.
[41] Maga E, Anderson G, Murray J. The effect of mammary gland expression of human lysozyme on the properities of milk from transgenic mice. Journal of Dairy Science.1995,78:2645 ~2652.
[42] Roberts B, Tullio P, Vitale J, et al.. Cloning of the goat betacasein-encoding gene and expression in transgenic mice. Gene.1992,121:255~262.
[43] Yu Z, Fan B, Li N, et al. Expression of recombinant human lysozyme in the milk of transgenic mice. Chinese Science Bulletin.2003, 48(21):2331~2335.
[44] 李国才, 孙怀昌, 孙强. 乳腺高效表达人溶菌酶转基因小鼠的制备.中华微生物学与免疫学杂志.2004,24(2):142~145.
[45] Murakami M, Jigani Y, Tanaka H, et al. Expression of synthetic human lysozyme gene in Escherichia coli. Agricultural Biological Chemistry.1986,(50):713~723.
[46] Yoshimura K, Toibana A, Nakahama K. Human lysozyme: Sequence of a cDNA, andexpression and secretion by Saccharomyces cerevisae. Biochemistry and Biophysics Research Communication.1988,(105):794～801.
[47] 贾向志, 袁汉英, 马文熠等. 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.
[48] 钱世钧, 陈军, 叶欣等. 从大肠杆菌包含体中提取有活性的人溶菌酶的研究. 生物工程学报.1996 ,12(增刊) :266～268.
[49] Fisher R, Emans N. Molecular farming of pharmaceutical proteins. Transgenic Research.2000,9:279~299.
[50] 焦瑞身. 展望即将到来的“分子农业”.生物工程学报.2001,17(4):361~364.
[51] Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants.Nature.1989,342:76~78.
[52] Ma J, Hein M. Immunotherpeuic potential of antibodies produced in plants. Trends Biotechnology.1995,13:522~527.
[53] Verwoerd T, Paridon P, Ooyen A. Stable accumulation of aspergillus niger phytase in transgenic tobacco leaves. Plant Physiology.1995,109:1199~1205.
[54] Magnuson N, Linzmaier P, Reeves R, et al. Secretion of biologically active human interleukin-2 and interleukin-4 from genetically modified tobacco cells in suspension culture. Protein Exprission and Purifiration.1998,13:45~52.
[55] Sijmons P, Dekker B, Schrammeijer B, et al. Production of correctly processed human serum albumin in transgenic plants. Biotechnology.1990,8:217~221.
[56] Walmsley A, Arntzen C. Plants for delivery of edible vaccines. Current Opinion in Biotechnology.2000,11:126~129.
[57] Fischer R, Stoger E, Schillberg S, et al. Plant-based production of biopharmaceuticals. Current Opinion in Plant Biology.2004,7(2):152~158.
[58] Walsh G. Pharmaceuticals, biologics and biopharmaceuticals. Biopharmaceuticals, Biochemistry and Biotechnology.Wiley, Chichester,UK.1998:1~35.
[59] 邱信芳. 人凝血因子 IX 基因乳腺特异表达载体的构建及其在奶山羊乳腺中的分泌性表达.生物工程学报.1997, 2:78~83.
[60] Echelard Y. Recombinant protein production in transgenic animals. Current Opinion in Biotechnology.1996,7(5):536~540.
[61] Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants. Nature.1989,342:76~78.
[62] Voss A, Niersbach M, Hain R, et al. Reduced virus infectivity in N. tabacum secreting a MV-specific full size antibody. Molecular Breeding.1995,1:39~50.
[63] Cabanes M, Fitchette A, Loutelier B, et al. N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants. Glycobiology.1999, 9:365~372.
[64] Ohya K, Itchoda N, Ohashi K, et al. Expression of biologically active human tumor necrosis factor~alpha in transgenic potato plant. Interferon Cytokine Research.2002,22(3):371~378.
[65] Ziegler M, Thomas S, Danna K. Accumulation of a thermostable endo-1, 4-b-D-glucanase in the apoplast of Arabidopsis thaliana leaves. Molecular Breedding. 2000,6:37~41.
[66] Sambrook J, Fritsch F, Maniatis T. Molecular Cloning: A laboratory manual.3rd ed. New York: Cold Spring Harbor Laboratory.
[67] Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Analysis Biochemistry.1976,72(1~2):248~254.
[68] 苍金荣 , 刘万里 , 刘延龄等 . 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.
[69] 王关林, 方宏筠.植物基因工程.北京:科学出版社.2002,8:397~400.
[70] Lin R, Ding Z, Kuang T, et al. Plant Molecular Biology.2001,19:379a~379e
[71] Andersson S and Kurland C, Codon preferences in free-living microorganisms. Microbiology Review.1990,54(2):198~210.
[72] Mason H, Haq T, Clements J, et al. Edible vaccine protects mice against E.coli heat-labile enterotoxin: potatoes expressing a synthetic LT-B gene.Vaccine.1998,16(13):1336~1343.
[73] Julian K, Drake P. The procuction of recombinant pharmaceutical proteins in plants. Genetics.2003,4:794~805.
[74] Matthews P, Wang M, Waterhouse P, et al. Marker gene elimination from transgenic barley, using co-transformation with adjacent ?twin T-DNAs? on a standard Agrobacterium transformation vector. Molecular Breeding. 2001,7:195~202.
[75] Dale E, Ow D. Gene transfer with subsequent removal of the selection gene from the plant genome. Proceedings of the National Academy of Sciences.1991,88:10558~10562.
[76] Goldsbrough A, Lastrella C, Yoder J. Transposition mediated repositioning and subsequent elimination of marker genes from transgenic tomato.Biotechnology.1993,11:1286~1292.
[77] 王永飞, 马三梅, 亦如瀚. 转基因植物选择标记基因及其安全性问题.植物生理学通讯. 2004,40(5):604~610.
[78] Yoder J, Goldsbrough A. Transfomation systems for gerneration marker-free transgenic plants. Biotechnology.1994,12:263~267.
[79] De Block M, Debrouwer D. Two T-DNAs cotransformed into Brassica napus by a double Agrobacterium tumefaciens infection are mainly integrated at the same locus. Theory Applied Genetics.1991,82:257~263.
[80] Komari T, Hiei Y, Saito Y, et al. Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant Journal.1996,10:165~174.
[81] Lu H, Zhou X, Gong Z, et al. Generation of selectable marker-free transgenic rice using double right borders binary vectors. Plant Physiology.2001,28:241~248.
[82] Miller M, Tagliani L, Wang N, et al. High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system. Transgenic Research.2002,11:381~396.
[83] Frandsen G, Mundy J, Tzen J. Oil bodies and their associated proteins, oleosin and caleosin. Physiologia Plantarum.2001,112 (3):301~307.
[84] 刘 昱 辉 , 贾 士 荣 . 植 物 油 体 表 达 体 系 的 研 究 进 展 . 农 业 生 物 技 术 学报.2003,11(5):531~537.
[85] 曲勍, 李校堃, 于雅琴. 利用油体表达系统生产外源重组蛋白.中国生物工程杂志.2007,27(8):111～115.
[86] 陈琳, 张亚东. 嗜甲醇酵母表达系统研究进展.国外医学.2002,24(1):11~14.