人溶菌酶基因治疗奶牛乳腺炎的初步研究
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摘要
根据已发表的人溶菌酶(human lysozyme，hLYZ)mRNA序列设计引物，以人乳腺第一链cDNA为模板，用PCR方法扩增出1.5 kb hLYZ双链cDNA，其推导的氨基酸序列与国外发表的从胎盘、巨噬细胞和结肠中克隆的hLYZ氨基酸序列同源性为100％，与从人组织细胞中克隆的hLYZ仅有1个氨基酸差异，但与从中国人胎盘中克隆的hLYZ具有6个氨基酸差异。将此cDNA亚克隆入真核表达载体pcDNA3和乳腺特异表达载体pCX中，用所获得的基因构件pcDNA3LYZ转染COS-1细胞进行暂态表达，用免疫荧光法检测到表达产物；用基因构件pCXLYZ注射哺乳期小鼠，经微球菌溶解试验证明，上述基因构件能有效地驱动目的基因在小鼠乳腺中表达，表达量为69.3μg／ml。
将序列正确的hLYZ cDNA克隆入乳腺特异表达载体p205C3，用获得的重组质粒p205C3LYZ注射正常奶牛乳腺，通过微球菌琼脂扩散法检测其乳汁中溶菌酶含量达1.18-1.96 μ g／ml，表达至少维持8天。3次共注射600 μ g重组质粒于患隐性乳腺炎的奶牛乳腺中，于注射后第2、6、10天分别采集奶样，通过奶样的C．M．T变化来判断治疗效果，其有效率分别为4／14、14／14、12／14。对比治疗试验显示，基因治疗和抗菌素治疗的有效率分别为86.4％(19／22)和88.9％(8／9)，显著高于中药治疗组的有效率(69.2％，9／13)。治疗有效的奶牛乳腺炎症状消失，葡萄球菌、链球菌和大肠杆菌及细菌总数明显减少，奶牛日均产奶量显著增高。用重组质粒对抗菌素治疗无效的18例临床型乳腺炎进行治疗试验，经一疗程治疗后除一例无效外，其余奶牛乳腺炎的红、肿、热、痛症状消失，乳汁颜色恢复正常。试验结果表明，含hLYZ cDNA的重组质粒p205C3LYZ对于奶牛乳腺炎具有良好的治疗效果。
Dairy cow mastitis is one of the most harmful and common cow diseases. It causes great loss every year in the world. Because of the complexity of the causative bacteria, dairy cow mastitis was very difficult to treat.
To obtain a functional cDNA for human lysozyme (hLYZ), PCR was carried out using the pooled first-stranded cDNA from human mammary gland as the template and 1.5 kb dscDNA was amplified by PCR. The cDNA was subcloned into pGEM-T vector and submitted to sequence analysis. The deduced amino acid sequence alignment showed that the cDNA was 100% identical to that cloned from human placenta, macrophages and colon, and differed by 1 and 6 amino acids from that cloned from histiocytes and Chinese placenta, respectively. The cDNA was subcloned into one eukaryotic expression vector and one mammary gland-specific expression vector. The recombinant vector, pcDNASLYZ, was transfected into COS-1 cells following mixing with branched 25 kDa polyethylenimine. Expression of hLYZ was revealed by indirect immunofluorecemce using specific antibody against hLYZ. Another recombinant vector, pCXLYZ, was injected into lactational mice via tail vein route. Expression of hLYZ at level of 69.3μg/ml was detected in the milk samples from
the gene-injected mouse using micrococcal lysis assay.
To treat cow mastitis by gene therapy, the hLYZ cDNA was subcloned into self-constructed mammary gland-specific expression vector p205C3 and the recombinant vector p205C3LYZ was injected into milk pools of the mammary gland. The hLYZ activity at level of >1.18μg/ml was detected in the milk samples from the plasmid-injected mammary gland, which maintained for at least 8 days. In treatment 1, 3x200μg of p205C3LYZ was injected into the milk pools of cows with mastitis and the effectiveness of the treatment was evaluated by C.M.T. of the milk samples, with the efficiency of 4 714, 14 714, 12/14 at the 2nd, 6th and 10th day post injection, respectively. In treatment 2, the effectiveness of gene therapy was compared with that of antibiotics treatment and Chinese herbs treatment, which showed that gene therapy and antibiotics treatment had an effectiveness of 84.6%(19/22) and 88.9%(8/9), respectively, both of which were significantly higher than that of ruyanxiao (69.2%,
9/13), a preparation of Chinese herbs. After the treatments, total bacterial numbers, as well as representative causative bacterial numbers (such as staphylococcus, streptococcus and E.coli), of the samples of the three groups decreased significantly. The milk yields of the three treatment groups increased significantly during and post treatment compared to pretreatments. The clinical cow mastitis was treated by the same way with the effectiveness of 55.6%(10/18). After treatment, the milk recovered to normal color and the clinical syndrome disappeared. These dada demonstrate that the recombinant plasmid containing hLYZ cDNA can be used as a new drug for preventing and curing dairy cow mastitis.
将序列正确的hLYZ cDNA克隆入乳腺特异表达载体p205C3，用获得的重组质粒p205C3LYZ注射正常奶牛乳腺，通过微球菌琼脂扩散法检测其乳汁中溶菌酶含量达1.18-1.96 μ g／ml，表达至少维持8天。3次共注射600 μ g重组质粒于患隐性乳腺炎的奶牛乳腺中，于注射后第2、6、10天分别采集奶样，通过奶样的C．M．T变化来判断治疗效果，其有效率分别为4／14、14／14、12／14。对比治疗试验显示，基因治疗和抗菌素治疗的有效率分别为86.4％(19／22)和88.9％(8／9)，显著高于中药治疗组的有效率(69.2％，9／13)。治疗有效的奶牛乳腺炎症状消失，葡萄球菌、链球菌和大肠杆菌及细菌总数明显减少，奶牛日均产奶量显著增高。用重组质粒对抗菌素治疗无效的18例临床型乳腺炎进行治疗试验，经一疗程治疗后除一例无效外，其余奶牛乳腺炎的红、肿、热、痛症状消失，乳汁颜色恢复正常。试验结果表明，含hLYZ cDNA的重组质粒p205C3LYZ对于奶牛乳腺炎具有良好的治疗效果。
Dairy cow mastitis is one of the most harmful and common cow diseases. It causes great loss every year in the world. Because of the complexity of the causative bacteria, dairy cow mastitis was very difficult to treat.
To obtain a functional cDNA for human lysozyme (hLYZ), PCR was carried out using the pooled first-stranded cDNA from human mammary gland as the template and 1.5 kb dscDNA was amplified by PCR. The cDNA was subcloned into pGEM-T vector and submitted to sequence analysis. The deduced amino acid sequence alignment showed that the cDNA was 100% identical to that cloned from human placenta, macrophages and colon, and differed by 1 and 6 amino acids from that cloned from histiocytes and Chinese placenta, respectively. The cDNA was subcloned into one eukaryotic expression vector and one mammary gland-specific expression vector. The recombinant vector, pcDNASLYZ, was transfected into COS-1 cells following mixing with branched 25 kDa polyethylenimine. Expression of hLYZ was revealed by indirect immunofluorecemce using specific antibody against hLYZ. Another recombinant vector, pCXLYZ, was injected into lactational mice via tail vein route. Expression of hLYZ at level of 69.3μg/ml was detected in the milk samples from
the gene-injected mouse using micrococcal lysis assay.
To treat cow mastitis by gene therapy, the hLYZ cDNA was subcloned into self-constructed mammary gland-specific expression vector p205C3 and the recombinant vector p205C3LYZ was injected into milk pools of the mammary gland. The hLYZ activity at level of >1.18μg/ml was detected in the milk samples from the plasmid-injected mammary gland, which maintained for at least 8 days. In treatment 1, 3x200μg of p205C3LYZ was injected into the milk pools of cows with mastitis and the effectiveness of the treatment was evaluated by C.M.T. of the milk samples, with the efficiency of 4 714, 14 714, 12/14 at the 2nd, 6th and 10th day post injection, respectively. In treatment 2, the effectiveness of gene therapy was compared with that of antibiotics treatment and Chinese herbs treatment, which showed that gene therapy and antibiotics treatment had an effectiveness of 84.6%(19/22) and 88.9%(8/9), respectively, both of which were significantly higher than that of ruyanxiao (69.2%,
9/13), a preparation of Chinese herbs. After the treatments, total bacterial numbers, as well as representative causative bacterial numbers (such as staphylococcus, streptococcus and E.coli), of the samples of the three groups decreased significantly. The milk yields of the three treatment groups increased significantly during and post treatment compared to pretreatments. The clinical cow mastitis was treated by the same way with the effectiveness of 55.6%(10/18). After treatment, the milk recovered to normal color and the clinical syndrome disappeared. These dada demonstrate that the recombinant plasmid containing hLYZ cDNA can be used as a new drug for preventing and curing dairy cow mastitis.
引文
1 Lehninger AL, Nelson DL, Cox MM. Principles of Biochemistry. 2nd Edition. New York: Worth Publishers. 1993:180-183.
2 张凤凯，马美湖．溶菌酶及其在食品保鲜剂的应用．肉类研究，2001，4：41-42．
3 谢宪章．天然食品防腐剂有待开发．食品科学，1995，12：6-8．
4 朱奇，陈彦．溶菌酶及其应用．生物学通报，1998，10：9-10．
5 方元超，梅丛笑，尹宁．溶菌酶及其应用前景．中国食品添加剂，1999，04：39-43．
6 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．
7 杨保全，许卫．溶菌酶在可腔疾病中的作用．现代可腔医学杂志，1990，4(1)：40-41．
8 史萸芳．溶菌酶抑菌作用的实验．牙体牙髓牙周病学杂志，1996，6(1)：26-28．
9 谢秩勋，刘雄等．热负荷对溶菌酶活性的影响．中国免疫学杂志，1997，13：148-149．
10 郇京宁等．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．
11 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): 487-492.
12 杨保全，王克群．溶菌酶治疗牙周炎36例疗效观察．口腔医学，1989，9(4)：21-22．
13 张天明，沈渤江．溶菌酶的临床应用．山东医药，1988，28(2)：3．
14 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．
15 刘仲敏等．溶菌酶及其在食品工业中的应用．食品与发酵工业，1995，5：80-83．
16 Jarrett J A. Mechanical milking and its relationship to mastitis [J]. Vet. Clih. NorthAm. (LargeAnim.Pract.), 1984,6: 349-360.
17 刘纯传，陈初茂等．乳炎清治疗奶牛临床型乳房炎疗效观察．中国兽医科技，1997年02期：38-40．
18 WilliamC，RebhumDVM著，赵德明，沈建忠主译，奶牛疾病学[M]．中国农业大学出版社，1999年，第一版．
19 杨章平，王健，丁焕峰等．奶牛隐性乳房炎发生规律的研究[J]．中国奶牛，1998(1)：18-20．
20 BushnellRB. Mycoplasma Mastitis[J], Vet. Clin. North. Am.(LargeAnim.Prac.), 1984, 6: 301-302.
21 潘耀谦，郭顺远，杨盛华．奶牛不同型隐性乳腺炎的病理形态学观察[J]．辽宁畜牧兽医，1996，5：2-4．
22 Lenhardt L. histtochemical study of mastiffs mammary gland in lactating cows[J]. Vet. Med-Czech. 1999, 44(40): 109-113.
23 Kawai K et al. Leatoferrin concentration in milk of bovine clinical mastitis [J]. Veterinary Research Communication. 1999, 23; 391-398.
24 Urech E et al. Changes in milk protein fraction as affected by subclinical mastiffs[J]. Journal of Dairy Science, 1999, 82; 2402-2411.
25 Buragohin J and Durra G N. Treatment of bovine subclinical mastitis. [J]. Indian Vet. J 1999, 76(7): 646-649.
26 Dalject Ch, et al. Studies on mycotic mastitis in cattle [J]. Indian Vet. J. 1999,76(11): 1043-1045.
27 Wollf J A et al. Direct gene transfer into mouse muscle in vivo. Science, 1990, 247: 1465-1468.
28 卢一凡，邓继先．乳腺直接注射质粒DNA的转基因暂时性表达研究进展．国外医学遗传学分册，1998，21(6)：310-312．
29 LuYi-fan et al. Expression of human G-CSF in mammary gland of mice by injection of plasmid DNA. Developmental and Reproductive Biology, 1997, 2: 29-32.
30 Lai Liang-xue et al. Expression of human tPA in mammary gland of rabbit by injection of plasmid DNA. Developmental and Reproductive Biology, 1999,
8(1)：17-23．
31 成勇，徐少甫等．牛aS1酪蛋白／HBsAg基因在山羊乳腺中暂态表达．扬州大学学报，1998，1(3)：27-31．
32 王贵，卢一凡等．乳腺直接注射重组质粒表达人G-CSF的研究．核农学报，1997，4：221．
33 Ilan N et al. Establishment and initial characterization of the ovine mammary epithelial cell line NISH. In Vitro Cell Dev Biol Anim, 1998, 34: 326-332.
34 李震，林爱星等．牛乳腺细胞系BME-L1 β-酪蛋白分泌的诱导及人促红细胞生成素(hEPO)的表达．农业生物技术学报，1999，7(1)：69-72．
35 张克忠，邱信芳．乳腺生物反应器的基础研究-载体构建，检测及反应器的建立方法．外医学遗传学分册，1997，20(6)：314-318．
36 俞小忠，马勇等．溶菌酶抗体的制备及其酶联免疫吸附试验的建立．中华医学检疫杂志，1998，01(21)：39．
37 Yoshimura K, Toibana A, Nakahama K. Human lysozyme: sequencing of a cDNA, and expression and secretion by Saccharomyces eerevisiae. Biochem. Biophys. Res. Commun. 1988, 150(2): 794-801.
38 姆布鲁克 J，弗里奇 EF，曼尼阿蒂斯 T．分子克隆实验指南．第2版．金冬雁，黎孟枫译．北京：科学出版社，1993．
39 鄂征．组织培养和分子细胞学技术．北京科技出版社，1995．
40 Sambrook J, Russell DW. Molecular Cloning A Laboratory Manual. Third Edition. New York: Cold Spring Harbor Laboratory Press. 2002.
41 曾林，付兴伦，贾世玉．家兔初乳溶菌酶含量测定．中国家兔杂志．1999，5：19-20．
42 姜秀云，吴惠毅．平板法测定血清溶菌酶活性影响因素探讨．上海医学检疫杂志，1994，9(1)：12．
43 Castanon MJ, Spevak W, Adolf GR, et al. Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae. Gene. 1988, 66: 223-234.
44 Chung LP, Keshav S, Gordon S, et al. 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.
45 Huang B, Zhao C, Lei X, etal. The cloning, sequencing and analysis of Chinese human lysozyme gene. CDNA amplified with RT-PCR from human placental total RNA. Chin. Bioehem. J. 1993, 9: 269-273.
46 Jolles J, Jolles P. Human milk lysozyme: un published data concerning the establishment of the complete primary structure; comparison with lysozymes of various origins. Helv. Chim. Acta. 1971, 54 (8): 2668-2675.
2 张凤凯，马美湖．溶菌酶及其在食品保鲜剂的应用．肉类研究，2001，4：41-42．
3 谢宪章．天然食品防腐剂有待开发．食品科学，1995，12：6-8．
4 朱奇，陈彦．溶菌酶及其应用．生物学通报，1998，10：9-10．
5 方元超，梅丛笑，尹宁．溶菌酶及其应用前景．中国食品添加剂，1999，04：39-43．
6 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．
7 杨保全，许卫．溶菌酶在可腔疾病中的作用．现代可腔医学杂志，1990，4(1)：40-41．
8 史萸芳．溶菌酶抑菌作用的实验．牙体牙髓牙周病学杂志，1996，6(1)：26-28．
9 谢秩勋，刘雄等．热负荷对溶菌酶活性的影响．中国免疫学杂志，1997，13：148-149．
10 郇京宁等．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．
11 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): 487-492.
12 杨保全，王克群．溶菌酶治疗牙周炎36例疗效观察．口腔医学，1989，9(4)：21-22．
13 张天明，沈渤江．溶菌酶的临床应用．山东医药，1988，28(2)：3．
14 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．
15 刘仲敏等．溶菌酶及其在食品工业中的应用．食品与发酵工业，1995，5：80-83．
16 Jarrett J A. Mechanical milking and its relationship to mastitis [J]. Vet. Clih. NorthAm. (LargeAnim.Pract.), 1984,6: 349-360.
17 刘纯传，陈初茂等．乳炎清治疗奶牛临床型乳房炎疗效观察．中国兽医科技，1997年02期：38-40．
18 WilliamC，RebhumDVM著，赵德明，沈建忠主译，奶牛疾病学[M]．中国农业大学出版社，1999年，第一版．
19 杨章平，王健，丁焕峰等．奶牛隐性乳房炎发生规律的研究[J]．中国奶牛，1998(1)：18-20．
20 BushnellRB. Mycoplasma Mastitis[J], Vet. Clin. North. Am.(LargeAnim.Prac.), 1984, 6: 301-302.
21 潘耀谦，郭顺远，杨盛华．奶牛不同型隐性乳腺炎的病理形态学观察[J]．辽宁畜牧兽医，1996，5：2-4．
22 Lenhardt L. histtochemical study of mastiffs mammary gland in lactating cows[J]. Vet. Med-Czech. 1999, 44(40): 109-113.
23 Kawai K et al. Leatoferrin concentration in milk of bovine clinical mastitis [J]. Veterinary Research Communication. 1999, 23; 391-398.
24 Urech E et al. Changes in milk protein fraction as affected by subclinical mastiffs[J]. Journal of Dairy Science, 1999, 82; 2402-2411.
25 Buragohin J and Durra G N. Treatment of bovine subclinical mastitis. [J]. Indian Vet. J 1999, 76(7): 646-649.
26 Dalject Ch, et al. Studies on mycotic mastitis in cattle [J]. Indian Vet. J. 1999,76(11): 1043-1045.
27 Wollf J A et al. Direct gene transfer into mouse muscle in vivo. Science, 1990, 247: 1465-1468.
28 卢一凡，邓继先．乳腺直接注射质粒DNA的转基因暂时性表达研究进展．国外医学遗传学分册，1998，21(6)：310-312．
29 LuYi-fan et al. Expression of human G-CSF in mammary gland of mice by injection of plasmid DNA. Developmental and Reproductive Biology, 1997, 2: 29-32.
30 Lai Liang-xue et al. Expression of human tPA in mammary gland of rabbit by injection of plasmid DNA. Developmental and Reproductive Biology, 1999,
8(1)：17-23．
31 成勇，徐少甫等．牛aS1酪蛋白／HBsAg基因在山羊乳腺中暂态表达．扬州大学学报，1998，1(3)：27-31．
32 王贵，卢一凡等．乳腺直接注射重组质粒表达人G-CSF的研究．核农学报，1997，4：221．
33 Ilan N et al. Establishment and initial characterization of the ovine mammary epithelial cell line NISH. In Vitro Cell Dev Biol Anim, 1998, 34: 326-332.
34 李震，林爱星等．牛乳腺细胞系BME-L1 β-酪蛋白分泌的诱导及人促红细胞生成素(hEPO)的表达．农业生物技术学报，1999，7(1)：69-72．
35 张克忠，邱信芳．乳腺生物反应器的基础研究-载体构建，检测及反应器的建立方法．外医学遗传学分册，1997，20(6)：314-318．
36 俞小忠，马勇等．溶菌酶抗体的制备及其酶联免疫吸附试验的建立．中华医学检疫杂志，1998，01(21)：39．
37 Yoshimura K, Toibana A, Nakahama K. Human lysozyme: sequencing of a cDNA, and expression and secretion by Saccharomyces eerevisiae. Biochem. Biophys. Res. Commun. 1988, 150(2): 794-801.
38 姆布鲁克 J，弗里奇 EF，曼尼阿蒂斯 T．分子克隆实验指南．第2版．金冬雁，黎孟枫译．北京：科学出版社，1993．
39 鄂征．组织培养和分子细胞学技术．北京科技出版社，1995．
40 Sambrook J, Russell DW. Molecular Cloning A Laboratory Manual. Third Edition. New York: Cold Spring Harbor Laboratory Press. 2002.
41 曾林，付兴伦，贾世玉．家兔初乳溶菌酶含量测定．中国家兔杂志．1999，5：19-20．
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