人溶菌酶动物乳腺生物反应器的研制
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摘要
人溶菌酶(human lysozyme，hLYZ)能水解细菌细胞壁粘多糖的β-1，4糖苷键，所以对革兰阳性细菌具有直接的溶解作用，在分泌型免疫球蛋白A和补体的参与下对革兰氏阴性细菌具有间接的溶解作用，因此在医学、食品工业等领域得到十分广泛的应用。
目前使用的溶菌酶主要从鸡蛋清中提取，天然的hLYZ不仅来源有限，而且提取方法复杂、价格昂贵。目前虽有细菌和酵母表达hLYZ的报道，但普遍存在转译后加工修饰不完全、溶解宿主细胞壁和表达水平低等问题，难以进行工厂化生产。转基因动物乳腺生物反应器具有表达水平高、表达产物活性强、易于纯化和无污染等优点，是大规模生产重组蛋白的新途径。
为了研制表达hLYZ的动物乳腺生物反应器，本研究根据已发表的hLYZ序列设计引物，以人乳腺第一链cDNA为模板，用PCR方法扩增出长1.5 kb hLYZ双链cDNA。测序结果显示，其序列与已发表的从人胎盘、巨噬细胞和结肠中克隆的人hLYZ完全一致，其中包括1个447 bp的阅读框和3′端反向重复的Alu序列。将此cDNA序列推导成氨基酸序列，并与GenBank中已发表的hLYZ氨基酸序列进行比较，结果与从人胎盘、巨噬细胞和结肠中克隆的hLYZ完全相同，与从人组织细胞中克隆的hLYZ仅有1个氨基酸差异(41位的异亮氨酸→蛋氨酸替换)，但与从中国人胎盘中克隆的hLYZ具有5个氨基酸差异，分别是第10、111和128位的颉氨酸→丙氨酸替换，124位的异亮氨酸→颉氨酸替换和136位的天门冬氨酰胺→天门冬氨酸替换。因为上述5个氨基酸替换具有一定的规律性(3个为颉氨酸→丙氨酸替换)，所以这种序列差异可能是不同人种之间的遗传差异，而不是克隆过程中产生的随机错误。
将上述hLYZ cDNA克隆入真核表达载体pcDNA3，用获得的重组质粒转染COS-1细胞，经间接免疫荧光试验证明目的基因能进行正确表达。再将此cDNA
扬州大学博士学位论文
克隆入自行构建的乳腺表达载体P205C3、国外引进的乳腺表达载体pBJ41和
PBCI，获得的重组质粒分别命名为P205C3LYZ、PBJLYZ和PBCLYZ，经多聚乙
酞亚胺包裹后通过尾静脉注射哺乳期小鼠，经微球菌溶解试验证明三种重组载体
不仅能在乳腺细胞中表达，而且分泌在乳汁中的重组酶分别达到87、69、60m叭。
将基因注射小鼠扑杀，取其12种组织提取总RNA，经Dot blotting检侧证明，三
种基因构件除在乳腺中表达外，在脾、肠或肾中也有一定的异位表达。这些试验
结果提示，自行构建的乳腺表达载体可以替代国外引进载体进行动物乳腺生物反
应器研究。
用显微注射法将重组表达载体p205C3LYz注射入小鼠受精卵，获得136只F0
代小鼠，用PCR和s。川出em bfotting分别检测到目的基因整合阳性鼠7只(2早5
占)和4只(1早3占);在soulthern blo伪泣g检测阳性的4只F。鼠中，1只母鼠为
hLYZ表达阴性，2只雄鼠与正常母鼠交配出生的F，代母鼠为hLYZ表达阴性，1
只雄鼠的4只F，代母鼠乳汁中表达的让止yZ分别为5 mg/L、75 mg几、175m叭
和200m留1;从F:代开始，对表达阳性鼠进行兄妹交配，经PCR检测证明转基因
的遗传符合孟德尔遗传规律;目前转基因纯合鼠已传到F7代，每代母鼠乳汁中的
rhl万z表达量基本稳定，最高表达量达750m叭;将其中的3只母鼠扑杀，取其
组织制备细胞裂解液和总RNA，经微球菌溶解试验和Dot blotting检测证明，目
的基因除在乳腺表达外，在脾脏和小肠也有一定的异位表达。
取转基因阳性鼠的乳汁进行Westem blotting分析，结果显示乳中表达的八止yZ
与人初乳中天然的LYZ具有相同的分子里;将适当稀释的转基因小鼠乳汁和人初
乳在60，C、72℃、85，C、100，C水浴中孵育l、3、5、10、15、20、30 min，经微
球菌溶解试验证明，比山YZ与天然址万Z具有相似的热稳定性;将乳样的pH调节
为2、4、5、7.4、9、10，37℃作用30 min后进行酶活性测定，结果表明r址万Z
与天然址万Z的pH活性范围相近;将不同的金属离子加入乳样中，然后进行酶活
性测定，结果证明比LYZ和正常址笼Z对所试金属离子具有相似的敏感性;通过
琼脂平板扩散法检测不同种类的细菌对迁止YZ和hLYZ的敏感性，检出的敏感菌
在37℃培养过程中每隔3On五n取定量混合液进行琼脂平板菌落计数和绘制抑菌曲
线，结果表明表达的rhl笼Z与正常址万Z具有相似的抑菌活性。
用重组载体P205C3LYZ注射山羊受精卵，第一批试验使用的供、受体羊分别
李国才:人溶菌防动物乳膝生物反应器的研制
为17和22只，由于胚胎移植后遇到羊痘病毒感染，导致部分羊死亡，怀孕率很
低(4瓜2)，经PCR检测证明出生的3只羔羊为外源基因整合阴性;第二批试验分
别使用供、受体羊巧和23只，胚胎移植后35一40天的怀孕率为14/23，尽管流产
和产死胎羊相对较多，但仍获得12只存活羔羊;经Dotbl。币ng法初步检测证明，
其中7只为外源基因整合阳性。
Human lysozyme (hLYZ, EC3.2.1.17) hydrolyzes the glycosidic B-1, 4 linkage between N-acetyhnuramic acid and N-acetylglucosamine of the peptidoglycan polymer in the bacterial cell wall. Because of the direct exposure of polypeptidoglycan on the outside of the cell wall, hLYZ can directly lyze Gram-positive bacteria and indirectly lyze Gram-negative bacteria in the presence of slgA or complements. Furthermore, hLYZ has many other activities such as diminishing inflammation, detumescence, repairing putrescence tissues, improving blood supply in local tissues, reducing pus and anti-virus.
hLYZ can be purified from human breast milk, neutrophils, placenta and urine of hemodialysis patients. Because of the limited supply and concerns over the potential risk of transmitting pathogens to the nursing infants, development of safer recombinant hLYZ is needed. Expression of rhLYZ in prokaryotic bacteria or yeast by utilizing gene-engineering techniques has some difficulties due to their lack of appropriate post-translational modifications and the potential lysis of the host cells. Animal mammary gland bioreactor is a novel alternative strategy for production of recombinant proteins with many advantages including high production capacities, easy purification, faithful translational modifications and authentic biological activities of the expressed products.
To develop animal mammary gland bioreactors expressing hLYZ, 1.5 kb dscDNA for hLYZ was amplified by PCR from pooled first-stranded cDNAs from human mammary gland and subcloned into pGEM-T vector. Sequence analysis showed that the PCR-amplified cDNA was identical to that cloned from human placenta, macrophages and colon. The cDNA included an open reading frame of 447 bps and a pair of Alu elements in reverse orientation at the 3' -noncoding region. The open reading frame
encoded a polypeptide of 130 amino acids, the first 18 amino acids of which were predicted to be signal pepeptide. The mature enzyme had a predicted molecular weight of 14.7 kDa and pI of 9.02. The deduced amino acid sequence of the cDNA was also identical to that cloned from human placenta, macrophages and colon and differed by 1 or 5 amino acids from that cloned from histiocytes or Chinese placenta.
The cDNA was subcloned into eukaryotic expression vector pcDNA3 and transfected into COS-1 cells following mixing with 25 kDa branched polyethylenimine(PEI). Expression of hLYZ cDNA was revealed by indirect immunofluorecence using hLYZ-specific antibody. The hLYZ cDNA was then subcloned into mammary gland-specific vectors p205C3, pBJ41 and pBCl. After mixed with 25 kDa PEI, the three recombinant expression vectors p205C3LYZ, pBJLYZ and pBCLYZ were injected into lactating mice via tail vein route. Micrococcal lysis assay of the collected milk samples showed that rhLYZ was efficiently expressed in the mammary glands and secreted into the milk with maximal concentrations of 87, 69 and 60 mg/L, respectively, for the three recombinant vectors. The expression of the three vectors was restricted to mammary glands with some degrees of ectopic expression in spleen, intestines and/or kidney among 12 different tissues tested by dot blotting and micrococcal lysis assay. These data indicate that the self-constructed mammary gland-specific vector p205C3 can replace the other two vectors obtained from abroad for development of animal mammary gland bioreactors.
The recombinant vector p205C3LYZ was used to generate transgenic mice by microinjection. A total of 136 F0 mice were obtained, of which 7 (2 5 ) and 4 (1 3 ) were showed to be transgenic by PCR and Southern blotting, respectively. Based on the micrococcal lysis assay of the milk samples, the only one female founder did not express the gene of interest. The three male founders were mated with normal mice and four female offspring of the only one male founder expressed hLYZ activities at levels of 5-200 mg/L. From the F1 generation on, the transgenic mice were mated with their sisters or brothers and a mouse colony was established with stable transmission and expre
目前使用的溶菌酶主要从鸡蛋清中提取，天然的hLYZ不仅来源有限，而且提取方法复杂、价格昂贵。目前虽有细菌和酵母表达hLYZ的报道，但普遍存在转译后加工修饰不完全、溶解宿主细胞壁和表达水平低等问题，难以进行工厂化生产。转基因动物乳腺生物反应器具有表达水平高、表达产物活性强、易于纯化和无污染等优点，是大规模生产重组蛋白的新途径。
为了研制表达hLYZ的动物乳腺生物反应器，本研究根据已发表的hLYZ序列设计引物，以人乳腺第一链cDNA为模板，用PCR方法扩增出长1.5 kb hLYZ双链cDNA。测序结果显示，其序列与已发表的从人胎盘、巨噬细胞和结肠中克隆的人hLYZ完全一致，其中包括1个447 bp的阅读框和3′端反向重复的Alu序列。将此cDNA序列推导成氨基酸序列，并与GenBank中已发表的hLYZ氨基酸序列进行比较，结果与从人胎盘、巨噬细胞和结肠中克隆的hLYZ完全相同，与从人组织细胞中克隆的hLYZ仅有1个氨基酸差异(41位的异亮氨酸→蛋氨酸替换)，但与从中国人胎盘中克隆的hLYZ具有5个氨基酸差异，分别是第10、111和128位的颉氨酸→丙氨酸替换，124位的异亮氨酸→颉氨酸替换和136位的天门冬氨酰胺→天门冬氨酸替换。因为上述5个氨基酸替换具有一定的规律性(3个为颉氨酸→丙氨酸替换)，所以这种序列差异可能是不同人种之间的遗传差异，而不是克隆过程中产生的随机错误。
将上述hLYZ cDNA克隆入真核表达载体pcDNA3，用获得的重组质粒转染COS-1细胞，经间接免疫荧光试验证明目的基因能进行正确表达。再将此cDNA
扬州大学博士学位论文
克隆入自行构建的乳腺表达载体P205C3、国外引进的乳腺表达载体pBJ41和
PBCI，获得的重组质粒分别命名为P205C3LYZ、PBJLYZ和PBCLYZ，经多聚乙
酞亚胺包裹后通过尾静脉注射哺乳期小鼠，经微球菌溶解试验证明三种重组载体
不仅能在乳腺细胞中表达，而且分泌在乳汁中的重组酶分别达到87、69、60m叭。
将基因注射小鼠扑杀，取其12种组织提取总RNA，经Dot blotting检侧证明，三
种基因构件除在乳腺中表达外，在脾、肠或肾中也有一定的异位表达。这些试验
结果提示，自行构建的乳腺表达载体可以替代国外引进载体进行动物乳腺生物反
应器研究。
用显微注射法将重组表达载体p205C3LYz注射入小鼠受精卵，获得136只F0
代小鼠，用PCR和s。川出em bfotting分别检测到目的基因整合阳性鼠7只(2早5
占)和4只(1早3占);在soulthern blo伪泣g检测阳性的4只F。鼠中，1只母鼠为
hLYZ表达阴性，2只雄鼠与正常母鼠交配出生的F，代母鼠为hLYZ表达阴性，1
只雄鼠的4只F，代母鼠乳汁中表达的让止yZ分别为5 mg/L、75 mg几、175m叭
和200m留1;从F:代开始，对表达阳性鼠进行兄妹交配，经PCR检测证明转基因
的遗传符合孟德尔遗传规律;目前转基因纯合鼠已传到F7代，每代母鼠乳汁中的
rhl万z表达量基本稳定，最高表达量达750m叭;将其中的3只母鼠扑杀，取其
组织制备细胞裂解液和总RNA，经微球菌溶解试验和Dot blotting检测证明，目
的基因除在乳腺表达外，在脾脏和小肠也有一定的异位表达。
取转基因阳性鼠的乳汁进行Westem blotting分析，结果显示乳中表达的八止yZ
与人初乳中天然的LYZ具有相同的分子里;将适当稀释的转基因小鼠乳汁和人初
乳在60，C、72℃、85，C、100，C水浴中孵育l、3、5、10、15、20、30 min，经微
球菌溶解试验证明，比山YZ与天然址万Z具有相似的热稳定性;将乳样的pH调节
为2、4、5、7.4、9、10，37℃作用30 min后进行酶活性测定，结果表明r址万Z
与天然址万Z的pH活性范围相近;将不同的金属离子加入乳样中，然后进行酶活
性测定，结果证明比LYZ和正常址笼Z对所试金属离子具有相似的敏感性;通过
琼脂平板扩散法检测不同种类的细菌对迁止YZ和hLYZ的敏感性，检出的敏感菌
在37℃培养过程中每隔3On五n取定量混合液进行琼脂平板菌落计数和绘制抑菌曲
线，结果表明表达的rhl笼Z与正常址万Z具有相似的抑菌活性。
用重组载体P205C3LYZ注射山羊受精卵，第一批试验使用的供、受体羊分别
李国才:人溶菌防动物乳膝生物反应器的研制
为17和22只，由于胚胎移植后遇到羊痘病毒感染，导致部分羊死亡，怀孕率很
低(4瓜2)，经PCR检测证明出生的3只羔羊为外源基因整合阴性;第二批试验分
别使用供、受体羊巧和23只，胚胎移植后35一40天的怀孕率为14/23，尽管流产
和产死胎羊相对较多，但仍获得12只存活羔羊;经Dotbl。币ng法初步检测证明，
其中7只为外源基因整合阳性。
Human lysozyme (hLYZ, EC3.2.1.17) hydrolyzes the glycosidic B-1, 4 linkage between N-acetyhnuramic acid and N-acetylglucosamine of the peptidoglycan polymer in the bacterial cell wall. Because of the direct exposure of polypeptidoglycan on the outside of the cell wall, hLYZ can directly lyze Gram-positive bacteria and indirectly lyze Gram-negative bacteria in the presence of slgA or complements. Furthermore, hLYZ has many other activities such as diminishing inflammation, detumescence, repairing putrescence tissues, improving blood supply in local tissues, reducing pus and anti-virus.
hLYZ can be purified from human breast milk, neutrophils, placenta and urine of hemodialysis patients. Because of the limited supply and concerns over the potential risk of transmitting pathogens to the nursing infants, development of safer recombinant hLYZ is needed. Expression of rhLYZ in prokaryotic bacteria or yeast by utilizing gene-engineering techniques has some difficulties due to their lack of appropriate post-translational modifications and the potential lysis of the host cells. Animal mammary gland bioreactor is a novel alternative strategy for production of recombinant proteins with many advantages including high production capacities, easy purification, faithful translational modifications and authentic biological activities of the expressed products.
To develop animal mammary gland bioreactors expressing hLYZ, 1.5 kb dscDNA for hLYZ was amplified by PCR from pooled first-stranded cDNAs from human mammary gland and subcloned into pGEM-T vector. Sequence analysis showed that the PCR-amplified cDNA was identical to that cloned from human placenta, macrophages and colon. The cDNA included an open reading frame of 447 bps and a pair of Alu elements in reverse orientation at the 3' -noncoding region. The open reading frame
encoded a polypeptide of 130 amino acids, the first 18 amino acids of which were predicted to be signal pepeptide. The mature enzyme had a predicted molecular weight of 14.7 kDa and pI of 9.02. The deduced amino acid sequence of the cDNA was also identical to that cloned from human placenta, macrophages and colon and differed by 1 or 5 amino acids from that cloned from histiocytes or Chinese placenta.
The cDNA was subcloned into eukaryotic expression vector pcDNA3 and transfected into COS-1 cells following mixing with 25 kDa branched polyethylenimine(PEI). Expression of hLYZ cDNA was revealed by indirect immunofluorecence using hLYZ-specific antibody. The hLYZ cDNA was then subcloned into mammary gland-specific vectors p205C3, pBJ41 and pBCl. After mixed with 25 kDa PEI, the three recombinant expression vectors p205C3LYZ, pBJLYZ and pBCLYZ were injected into lactating mice via tail vein route. Micrococcal lysis assay of the collected milk samples showed that rhLYZ was efficiently expressed in the mammary glands and secreted into the milk with maximal concentrations of 87, 69 and 60 mg/L, respectively, for the three recombinant vectors. The expression of the three vectors was restricted to mammary glands with some degrees of ectopic expression in spleen, intestines and/or kidney among 12 different tissues tested by dot blotting and micrococcal lysis assay. These data indicate that the self-constructed mammary gland-specific vector p205C3 can replace the other two vectors obtained from abroad for development of animal mammary gland bioreactors.
The recombinant vector p205C3LYZ was used to generate transgenic mice by microinjection. A total of 136 F0 mice were obtained, of which 7 (2 5 ) and 4 (1 3 ) were showed to be transgenic by PCR and Southern blotting, respectively. Based on the micrococcal lysis assay of the milk samples, the only one female founder did not express the gene of interest. The three male founders were mated with normal mice and four female offspring of the only one male founder expressed hLYZ activities at levels of 5-200 mg/L. From the F1 generation on, the transgenic mice were mated with their sisters or brothers and a mouse colony was established with stable transmission and expre
引文
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2 Blake CCF. Koening DF, Mair GA, et al. Structure of hen egg white lysozyme. Nature, 1965,206:757-764
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19 Maattheus BW, Crutter MG, Anderson WF, et al. Common precursor of lysozyme of hen egg-white and bacteriophage T4. Nature, 1981,290:334-345
20 Alexander J. Albreeht ES, Manuel G, et al. Exon encode functional and structural units of chicken lysozyme. Proc Nail Acad Sei USA, 1980, 77(10): 5759-5763
21 Lemos FJ, Terra WR. Digestion of bacteria and the role of midgut lysozyme in some larvae. Comp Bioche Physiol B, 1991, 100(2): 265-268
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23 Ford LD, Johnson LN, Machin PA, et al. Crystal structure of a lysozyme-tetrasaccharide lactone complex. J Mol Biol, 1974, 8:349-371
24 Sava G, Pacor S, Dasic G, et al. Lysozyme stimulates lymphocyte response to ConA IL-2 and potentates 5-fluorouracil action on advanced carcinomas. Anti cancer Res, 1995, 15:1883-1888
25 Lemarbe P. Lysozyme enhances monocyte-mediated tumoricidal activity: a potential amplifying mechanism of tumor killing. Blood, 1981, 58:994-999
26 Vieira J, Mssing J. The pUC plasmid a M13 mp7-derived system for insertion
matagenisis and sequencing with synthetic universal primers. Gene, 19:259-268
27 Stefan S, Michael B, Christopher MH, et al. An in vitro assay of b-galactosidase from yeast. Biotechniques, 1996, 20:960-962
28 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
29 Wu T, Samaranayake LP, Leung WK, et al. Inhibition of growth and secreted aspartyl proteinase production in Candida albieans by lysozyme. J Med Microbiol. 1999; 48(8): 721-30.
30 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
31 Pepys M B, Hawkins P N, Booth D R, et al. Human lysozyme gene mutations cause hereditary systemic amyloidosis. Nature, 1993, 362:553-557
32 Adriana I, Licz A, Laura U, et al. Determination of the fractal dimension of the lysozyme backbone of three different organisms. Chaos, Solitons and Fractals, 2001, 12:757-760
33 郁京宁，韩一平，陈玉林，等。溶葡萄球菌酶对烧伤小鼠吞噬细胞功能的影响。中华整形烧伤外科杂志，1995，11(4)：255-257
34 Krusteva E, Hristova S, Damyanov D, et al. Clinical study of the effect of the preparation DEODAN on leukopenia, induced by cytostatics. Int J Immunopharmacol. 1997; 19(9-10): 487-92.
35 Hisham R I, Tetsuji M, Takayoshi A, et al. Genetic evidence that antibacterial lactivity of lysozme is independent of its catalytic function. FEBS Letters, 2001, (506): 27-32
36 Shigeru M, Masato K, Masanori N, et al. Secretion of active human lysozyme by Acremonium chrysogenum using a Fusarium alkaline protease promoter system. J Biotech, 1995, (42): 1-8
37 刘仲敏，何伯安．溶菌酶及其在食品工业中的应用．食品与发酵工业，1995，(5)：80-82
38 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
39 Yuji Ito, Hidenori YAMADA, Taiji IMOTO. Colorimetric Assay for Lysozyme using Micrococcus luteus Labeled with a Blue Dye, Remazol Brilliant Blue, as a
Substrate. Chem. Pharm. Bull. 1992, 40(6): 1523-1526
40 吴晓英，林影，陈慧英．溶菌酶的研究进展．工业微生物，2002，32(4)：55-58
41 Strelets EV, Egorova EN, Chernin VV, et al. Properties and clinical application of quasi-liquid form of lysozyme Zh Mikrobiol Epidemiol Immunobiol. 2001; (3): 14-7.
42 Klaeger AJ, Cevallos V, Sherman MD, et al. Clinical application of a homogeneous colorimetric assay for tear lysozyme. Ocul Immunol Inflamm. 1999, 7(1): 7-15.
43 李冬梅．溶菌酶及在食品中的应用．食品工业，1999，3：21-22
44 Wang CS, Kloer HU. Purification of human lysozyme from milk and pancreatic juice. Anal Biochem. 1984; 139: 224- 227.
45 Lollike K, Kjeldsen L, Sengelon.H, et al. Purification of lysozyme from human neutrophils, and development of an ELISA for quantification in cells and plasma. Leukemia. 1995, 9:206-209
46 Takai I, Oda O, Shinzato T, et al. Effective method for purification of lysozyme from human urine. J Chromatogr B Biomed Appl. 1996; 685:21 - 25.
47 Barling PM, John M J, Walsh JR, et al The isolation and characterization of lysozyme from human foetal membranes: a comparison with the enzyme from other sources. Comp Biochem Physiol B. 1985; 81(2): 509-13.
48 Peters CW, Kruse U, Pollwein R, et al. The human lysozyme gene: Sequence organization and chromosomal localization. Eur J Biochem. 1989; 182(3): 507-516.
49 Riccio ML, Rossolini GM. Unusual clustering of Alu repeats within the 5'-flanking region of the human lysozyme gene. DNA Seq. 1993; 4(2): 129-134
50 Chung LP, Keshav S, Gordon S. Cloning the human lysozyme cDNA: inverted Alu repeat in the mRNA and in situ hybridization for macrophages and Paneth cells. Proc Natl Acad Sci U.S.A. 1988; 85(17): 6227-6231.
51 Yoshimura K, Toibana A, Nakahama K. Human lysozyme: sequencing of a cDNA, and expression and secretion by Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1988; 150(2): 794-801.
52 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)：266-265
53 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
54 Koshiba T, Tsumoto K, Masaki K, et al. Calorimetric study of mutant human lysozymes with partially introduced Ca~(2+) binding sites and its efficient refolding system from inclusion bodies. Protein Engng, 1998, 11, 683-690.
55 Castanon MJ, Spevak W, Adolf GR, et al. Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae. Gene. 1988; 66(2): 223-234.
56 Gregg JM. Madden KR, Bamhager KJ, et al. High-level expression and secreation of heterologous proteins from the methylotrophic yeast pichia pastoris. Mol Cell Biol. 1989, 9:1316-1320
57 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
58 Andrew S, Ludmilla AM, Wim N, et al. Expression, purification, and characterization of the recombinant calcium binding equine lysozyme secreted by the filamentous fungi Aspergillus niger: Comparisions with the production of hen and human lysozymes. Prot Expr Puri, 1999, (16): 171-180
59 王佃亮．重组人溶菌酶研究进展．中国生物工程杂志，2003，23(9)：59-63
60 Koganesawa N, Aizawa T, Masaki K, et al. Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system. Protein Eng. 2001, 14(9): 705-710.
61 刘德虎．利用转基因植物生产药用蛋白的研究现状．生物技术通报，1999，(4)：1-5
62 Nakajima H, Muranaka T, Ishige F. Fungal and bacterial disease resistance in transgenic plants expression human lysozyme. Plant Cell Report, 1994, 16:674-679
63 王冰山，窦道龙，张永强，等．人溶菌酶基因在烟草中的表达．农业生物技术学报，2002，10(4)：338-342
64 Jianmin Huang, Liying Wu, Dorice Yalda, et al. Expression of functional
recombinant human lysozyme in transgenic rice cell culture. Transgenic Research. 2002, 11: 229-239.
65 Jianmin Huang, Somen Nandi, Liying Wu, et al. Expression of natural antimicrobial human lysozyme in rice grains. Mole Bree, 2002, 10: 83-94.
66 Palmiter RD, Brinster RL, Hammer RE, et al. Dramatic growth of mice that develop from eggs microin-jected with metallothionein-growth hormone fusiongenes Nature. 1982; 300(5893): 611-615
67 Janne J, Alhonen L, Hyttinen JM, et al. Transgenic bioreactors. Biotechnol Annu Rev. 1998; 4: 55-74.
68 Hyttinen JM, Peura T, Tolvanen M, et al. Generation of transgenic dairy carle from transgenic analyzed and sexed embryos produced in vitro. Bio/Technology. 1994, 12:606-608
69 McEcoy TG, Sreensan JM. The efficiency of production, centrifugation, microinjection and transfer of one and two-cell bovine ova in a gene transfer program. Theriogenology, 1990, 3:819-828.
70 孙国杰，桑润滋，立俊杰，等．哺乳动物乳腺生物反应器的研究现状及存在问题．黄牛杂志，2002，28(3)：28-31
71 Larrick JW, Thomas DW. Producing proteins in transgenic plants and animals. Curr Opin Biotechnol. 2001, 12(4): 411-8.
72 Fan J, Watanabe T. Transgenic rabbits as therapeutic protein bioreactors and human disease models. Pharmacol Ther. 2003, 99(3): 261-82.
73 Maga EA, Anderson GB, Huang MC, et al. Expression of human lysozyme mRNA in the mammary gland of transgenic mice. Transgenic Res 1994; 3(1): 36-42
74 Maga EA, Anderson GB. The Effect of Mammary Gland Expression of Human Lysozyme on the Properties of Milk from Transgenic Mice. J Dairy Sci 1995, 78: 2645-2652
75 Maga EA, Anderson GB, Cullor JS, et al. Antimicrobial properties of human lysozyme transgenic mouse milk. J Food Prot. 1998; 61(1): 52-56.
76 于政权，樊宝良，戴蕴平，等．转基因小鼠乳腺表达重组人溶菌酶．科学通报，
2003，48(20)：2149-2153
77 孙怀昌，张泉，施伟庆，等．人溶菌酶cDNA的克隆及其在小鼠体内的表达．中国兽医学报，2004，24(2)：157-159
78 陈刚，施伟庆，吴梅娟，等．奶山羊β-乳球蛋白基因及其调控区的克隆与序列分析．江苏农业研究．2001，22(2)58-62
79 孙怀昌，陈刚，张磊，等．动物乳腺特异性表达载体的构建及其表达特性研究．扬州大学学报(农业与生命科学版)，2004，24(4)：1-4
80 孙怀昌，于峰，苏建华等．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
81 李国才，孙怀昌，孙强，等．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
82 Lee-Huang S, Huang PL, Sun Y, et al. Lysozyme and RNases as anti-HIV components in beta-core preparations of human chorionic gonadotropin. Proc Nail Acad Sci USA 1999, 96: 2678-2681.
2 Blake CCF. Koening DF, Mair GA, et al. Structure of hen egg white lysozyme. Nature, 1965,206:757-764
3 Fogaca AC, da Silva PI Jr, Miranda MT, et al. Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus. J Biol Chem. 1999; 274(36): 25330-25334.
4 Podboronov VM. Comparative characteristics of lysozymes of different origin. Antibiot Khimioter. 1990; 35(8): 22-25.
5 (日)船津胜鹤，大典编著，李兴福译，荆永志校，溶菌酶，山东科学技术出版社，1982
6 Grutter MG, Weaver LH, Matthews BW. Goose lysozyme structure: an evolutionary link between hen and bacteriophage lysozymes. Nature. 1983; 303(5920): 828-831.
7 Weaver LH, Grutter MG, Remington SJ, et al. 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. J Mol Evol. 1984-85; 21(2): 97-111.
8 Isabelle VS, Monique D. Electrotransfer of basic proteins from nodenaturing polyacrylamide acid gels to nitrocellulose: detection of enzymatic and inhibitory activities and retertion of protein antigenicity, Anal Biochem, 1990, 186(2): 306-311
9 朱奇，陈彦．溶菌酶及其应用．生物学通报，1998，33(10)：9-10
10 Prager EM, Wilson AC, Arnheim N, et al. Widespread Distribution of lyszyme g in egg white of birds, J Biol Chem, 1974, 249:7295-7297
11 Jolles P, Jolles J, What is new in lysozyme research? Always a model system, today as yesterday. Mol Cell Biol Chen, 1984, 63:165-189
12 Schoentgen F, Jolles J, Jolles P, et al. Complete amino acid sequence of ostrich
egg-white lysozyme, a goose-type lysozyme, Eur J biochem, 1982, 123: 489-497.
13 Goto NK, Skrynnikov NR, Dahlquist FW, et al. What is the average conformation of bacteriophage T4 lysozyme in solution? A domain orientation study using dipolar couplings measured by solution NMR. J Mol Biol. 2001; 308(4): 745-64.
14 Matthews BW. Structural and genetic analysis of the folding and function of T4 lysozyme. FASEB J. 1996; 10(1): 35-41.
15 Fastrez J. Phage lysozymes. EXS. 1996; 75: 35-64.
16 Inouye M, Imada M, Tsugita A, et al, The amino acid sequence of T4 phage lysozyme. Ⅰ. Tryptic digestion.. J Biol Chem, 1970, 245:3439-3454
17 Inouye M, Imada M, Akaboshi E, et al. The amino acid sequence of T4 phage lysozyme. Ⅱ. Chymotryptic digestion. J Biol Chem. 1970; 245(14): 3455-66.
18 Matthews BW, Remington SJ, Grutter MG, et al. Relation between hen egg white lysozyme and bacteriophage T4 lysozyme: evolutionary implications. J Mol Biol. 1981; 147(4): 545-58.
19 Maattheus BW, Crutter MG, Anderson WF, et al. Common precursor of lysozyme of hen egg-white and bacteriophage T4. Nature, 1981,290:334-345
20 Alexander J. Albreeht ES, Manuel G, et al. Exon encode functional and structural units of chicken lysozyme. Proc Nail Acad Sei USA, 1980, 77(10): 5759-5763
21 Lemos FJ, Terra WR. Digestion of bacteria and the role of midgut lysozyme in some larvae. Comp Bioche Physiol B, 1991, 100(2): 265-268
22 贾向志，李元，马文煜．溶菌酶的研究进展．生物技术通报．2002，13(5)：374-377
23 Ford LD, Johnson LN, Machin PA, et al. Crystal structure of a lysozyme-tetrasaccharide lactone complex. J Mol Biol, 1974, 8:349-371
24 Sava G, Pacor S, Dasic G, et al. Lysozyme stimulates lymphocyte response to ConA IL-2 and potentates 5-fluorouracil action on advanced carcinomas. Anti cancer Res, 1995, 15:1883-1888
25 Lemarbe P. Lysozyme enhances monocyte-mediated tumoricidal activity: a potential amplifying mechanism of tumor killing. Blood, 1981, 58:994-999
26 Vieira J, Mssing J. The pUC plasmid a M13 mp7-derived system for insertion
matagenisis and sequencing with synthetic universal primers. Gene, 19:259-268
27 Stefan S, Michael B, Christopher MH, et al. An in vitro assay of b-galactosidase from yeast. Biotechniques, 1996, 20:960-962
28 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
29 Wu T, Samaranayake LP, Leung WK, et al. Inhibition of growth and secreted aspartyl proteinase production in Candida albieans by lysozyme. J Med Microbiol. 1999; 48(8): 721-30.
30 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
31 Pepys M B, Hawkins P N, Booth D R, et al. Human lysozyme gene mutations cause hereditary systemic amyloidosis. Nature, 1993, 362:553-557
32 Adriana I, Licz A, Laura U, et al. Determination of the fractal dimension of the lysozyme backbone of three different organisms. Chaos, Solitons and Fractals, 2001, 12:757-760
33 郁京宁，韩一平，陈玉林，等。溶葡萄球菌酶对烧伤小鼠吞噬细胞功能的影响。中华整形烧伤外科杂志，1995，11(4)：255-257
34 Krusteva E, Hristova S, Damyanov D, et al. Clinical study of the effect of the preparation DEODAN on leukopenia, induced by cytostatics. Int J Immunopharmacol. 1997; 19(9-10): 487-92.
35 Hisham R I, Tetsuji M, Takayoshi A, et al. Genetic evidence that antibacterial lactivity of lysozme is independent of its catalytic function. FEBS Letters, 2001, (506): 27-32
36 Shigeru M, Masato K, Masanori N, et al. Secretion of active human lysozyme by Acremonium chrysogenum using a Fusarium alkaline protease promoter system. J Biotech, 1995, (42): 1-8
37 刘仲敏，何伯安．溶菌酶及其在食品工业中的应用．食品与发酵工业，1995，(5)：80-82
38 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
39 Yuji Ito, Hidenori YAMADA, Taiji IMOTO. Colorimetric Assay for Lysozyme using Micrococcus luteus Labeled with a Blue Dye, Remazol Brilliant Blue, as a
Substrate. Chem. Pharm. Bull. 1992, 40(6): 1523-1526
40 吴晓英，林影，陈慧英．溶菌酶的研究进展．工业微生物，2002，32(4)：55-58
41 Strelets EV, Egorova EN, Chernin VV, et al. Properties and clinical application of quasi-liquid form of lysozyme Zh Mikrobiol Epidemiol Immunobiol. 2001; (3): 14-7.
42 Klaeger AJ, Cevallos V, Sherman MD, et al. Clinical application of a homogeneous colorimetric assay for tear lysozyme. Ocul Immunol Inflamm. 1999, 7(1): 7-15.
43 李冬梅．溶菌酶及在食品中的应用．食品工业，1999，3：21-22
44 Wang CS, Kloer HU. Purification of human lysozyme from milk and pancreatic juice. Anal Biochem. 1984; 139: 224- 227.
45 Lollike K, Kjeldsen L, Sengelon.H, et al. Purification of lysozyme from human neutrophils, and development of an ELISA for quantification in cells and plasma. Leukemia. 1995, 9:206-209
46 Takai I, Oda O, Shinzato T, et al. Effective method for purification of lysozyme from human urine. J Chromatogr B Biomed Appl. 1996; 685:21 - 25.
47 Barling PM, John M J, Walsh JR, et al The isolation and characterization of lysozyme from human foetal membranes: a comparison with the enzyme from other sources. Comp Biochem Physiol B. 1985; 81(2): 509-13.
48 Peters CW, Kruse U, Pollwein R, et al. The human lysozyme gene: Sequence organization and chromosomal localization. Eur J Biochem. 1989; 182(3): 507-516.
49 Riccio ML, Rossolini GM. Unusual clustering of Alu repeats within the 5'-flanking region of the human lysozyme gene. DNA Seq. 1993; 4(2): 129-134
50 Chung LP, Keshav S, Gordon S. Cloning the human lysozyme cDNA: inverted Alu repeat in the mRNA and in situ hybridization for macrophages and Paneth cells. Proc Natl Acad Sci U.S.A. 1988; 85(17): 6227-6231.
51 Yoshimura K, Toibana A, Nakahama K. Human lysozyme: sequencing of a cDNA, and expression and secretion by Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1988; 150(2): 794-801.
52 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)：266-265
53 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
54 Koshiba T, Tsumoto K, Masaki K, et al. Calorimetric study of mutant human lysozymes with partially introduced Ca~(2+) binding sites and its efficient refolding system from inclusion bodies. Protein Engng, 1998, 11, 683-690.
55 Castanon MJ, Spevak W, Adolf GR, et al. Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae. Gene. 1988; 66(2): 223-234.
56 Gregg JM. Madden KR, Bamhager KJ, et al. High-level expression and secreation of heterologous proteins from the methylotrophic yeast pichia pastoris. Mol Cell Biol. 1989, 9:1316-1320
57 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
58 Andrew S, Ludmilla AM, Wim N, et al. Expression, purification, and characterization of the recombinant calcium binding equine lysozyme secreted by the filamentous fungi Aspergillus niger: Comparisions with the production of hen and human lysozymes. Prot Expr Puri, 1999, (16): 171-180
59 王佃亮．重组人溶菌酶研究进展．中国生物工程杂志，2003，23(9)：59-63
60 Koganesawa N, Aizawa T, Masaki K, et al. Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system. Protein Eng. 2001, 14(9): 705-710.
61 刘德虎．利用转基因植物生产药用蛋白的研究现状．生物技术通报，1999，(4)：1-5
62 Nakajima H, Muranaka T, Ishige F. Fungal and bacterial disease resistance in transgenic plants expression human lysozyme. Plant Cell Report, 1994, 16:674-679
63 王冰山，窦道龙，张永强，等．人溶菌酶基因在烟草中的表达．农业生物技术学报，2002，10(4)：338-342
64 Jianmin Huang, Liying Wu, Dorice Yalda, et al. Expression of functional
recombinant human lysozyme in transgenic rice cell culture. Transgenic Research. 2002, 11: 229-239.
65 Jianmin Huang, Somen Nandi, Liying Wu, et al. Expression of natural antimicrobial human lysozyme in rice grains. Mole Bree, 2002, 10: 83-94.
66 Palmiter RD, Brinster RL, Hammer RE, et al. Dramatic growth of mice that develop from eggs microin-jected with metallothionein-growth hormone fusiongenes Nature. 1982; 300(5893): 611-615
67 Janne J, Alhonen L, Hyttinen JM, et al. Transgenic bioreactors. Biotechnol Annu Rev. 1998; 4: 55-74.
68 Hyttinen JM, Peura T, Tolvanen M, et al. Generation of transgenic dairy carle from transgenic analyzed and sexed embryos produced in vitro. Bio/Technology. 1994, 12:606-608
69 McEcoy TG, Sreensan JM. The efficiency of production, centrifugation, microinjection and transfer of one and two-cell bovine ova in a gene transfer program. Theriogenology, 1990, 3:819-828.
70 孙国杰，桑润滋，立俊杰，等．哺乳动物乳腺生物反应器的研究现状及存在问题．黄牛杂志，2002，28(3)：28-31
71 Larrick JW, Thomas DW. Producing proteins in transgenic plants and animals. Curr Opin Biotechnol. 2001, 12(4): 411-8.
72 Fan J, Watanabe T. Transgenic rabbits as therapeutic protein bioreactors and human disease models. Pharmacol Ther. 2003, 99(3): 261-82.
73 Maga EA, Anderson GB, Huang MC, et al. Expression of human lysozyme mRNA in the mammary gland of transgenic mice. Transgenic Res 1994; 3(1): 36-42
74 Maga EA, Anderson GB. The Effect of Mammary Gland Expression of Human Lysozyme on the Properties of Milk from Transgenic Mice. J Dairy Sci 1995, 78: 2645-2652
75 Maga EA, Anderson GB, Cullor JS, et al. Antimicrobial properties of human lysozyme transgenic mouse milk. J Food Prot. 1998; 61(1): 52-56.
76 于政权，樊宝良，戴蕴平，等．转基因小鼠乳腺表达重组人溶菌酶．科学通报，
2003，48(20)：2149-2153
77 孙怀昌，张泉，施伟庆，等．人溶菌酶cDNA的克隆及其在小鼠体内的表达．中国兽医学报，2004，24(2)：157-159
78 陈刚，施伟庆，吴梅娟，等．奶山羊β-乳球蛋白基因及其调控区的克隆与序列分析．江苏农业研究．2001，22(2)58-62
79 孙怀昌，陈刚，张磊，等．动物乳腺特异性表达载体的构建及其表达特性研究．扬州大学学报(农业与生命科学版)，2004，24(4)：1-4
80 孙怀昌，于峰，苏建华等．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
81 李国才，孙怀昌，孙强，等．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
82 Lee-Huang S, Huang PL, Sun Y, et al. Lysozyme and RNases as anti-HIV components in beta-core preparations of human chorionic gonadotropin. Proc Nail Acad Sci USA 1999, 96: 2678-2681.