抗GbpA-GBD单克隆抗体的制备及特性研究
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摘要
龋病是三大非传染性疾病之一，发病广，危害大。变形链球菌族(MS)是目前公认的致龋菌，其中与人类龋病发生关系密切的是变形链球菌(S.mutans)和远缘链球菌(S.sobrinu，又称茸毛链球菌)。MS在牙面的粘附、增殖是其致龋的基础。目前防龋研究主要围绕链球菌在牙面的粘附、聚集及增殖几个环节进行，介导MS在牙面粘附、聚集的毒力因子主要有表面蛋白抗原Ⅰ/Ⅱ(AgⅠ/Ⅱ)、葡糖基转移酶(GTFs)及葡聚糖结合蛋白(Gbps)。AgⅠ/Ⅱ、GTFs及GbpB在免疫防龋中具有明显的保护性防龋作用，GbpA在氨基酸序列上与GTFs的葡聚糖结合区(GBD)高度同源，但其GBD在被动免疫防龋中的作用尚未见报道。由于被动免疫避免了主动免疫时可能出现的与宿主组织的交叉反应及其它副作用，直接作用于口腔，因此易被人们接受，受到越来越多的关注。
第四军医大学硕士学位论文
一
本研究分为两个部分：
一、分泌抗GbPAGBD单克隆抗体细胞系的建立
主要实验内容包括：
1．杂交瘤细胞系的建立：以GBD为抗原，免疫Balb／c ／J’鼠，通过B淋巴
细胞杂交瘤技术，即用B淋巴细胞与骨髓瘤细胞驼／0进行细胞融合，经
筛选和克隆化后，间接ELISA法检测杂交瘤培养上清中有特异性抗体分泌
后，建立杂交瘤细胞系。
2．杂交瘤细胞特性分析：用秋水仙素阻抑法对杂交瘤细胞特性进行染色
体数目分析。
3．效价测定及Ig类别鉴定：用琼脂糖凝胶双向扩散法检测抗体亚类。并
检测腹水效价。
4．单克隆抗体的纯化：用饱和硫酸铰盐析法，对单克隆抗体进行纯化。先
后共融合2次，克隆生长率为100％，阳性率为35％，经过2上次克隆化，
获得了 6株稳定分泌抗GBD的杂交瘤细胞系，分别命名为 2C9jC5J。
3H6、4C9和4FIO，在体外连续培养传代，液氮冻存半年以上，复苏后生
长良好，分泌抗体性能稳定。染色体数目分析结果显示：2C9染色体数为
107．6土3．4条，3C5为 102．2土2．l条，3GI、3H6、4C9和4F10分别为 106．8
i 2．2、102．4 i 3．6、107．ZIZ．7、105．6 i 3．3。说明各细胞系均为融合细胞。
5．ELISA检测抗体效价，六株McAh的腹水效价均为 IX10“‘。2C9、3C5。
3GI和 4C9亚型为 IgGI，。链；3H6和 4F10亚型为 IgGZa，K链。
3
第四军医大学硕士学位论文
二、抗GbpA＊BD单克隆抗体特异性鉴定及相应抗原初步分析
1单克隆抗体的亲和常数：采用间接ELISA法测定MCAb亲和常数。
2 GBD单克隆抗体 Western blot免疫印迹杂交
3免疫组织化学染色
从亲和常数可知 3C5、3H6和 4F10的亲和力大于 2C9、4C9和 3GI的亲
和力。Western blot免疫印迹杂交结果显示，3C5、3H6和 4F10在分子量 43KD
处出现明显的兔疫反应条带，而阴性对照组、空白对照组无阳性条带，2C9。
4C9和3GI在43KD处出现弱阳性条带。至少有3株单抗能够识别变性的
GBD蛋白，证实了单抗的特异性，同时也表明此6株单抗识别空间表位有
所差异。免疫组织化学染色检测结果发现，6株单抗在S，nutans胞壁染色均
呈阳性，进一步证实了其特异性。
Dental caries is one of the three chief non-contagious diseases which threaten human health. Mutans streptococci (MS) are the primary cariogenic agents, and Streptococcus mutans and Streptococcus sobrinus are believed to have the closest relationship with human caries. Adhesion and proliferation of MS are the basis of cariogensis. At present, the study of anticaries mainly focuses on a series of events involving the adherence, accumulation and proliferation of MS on tooth surfaces. Researchers have done many studies on cloning, functions and anticaries immunization of the genes related to adherence and accumulation. These genes includ surface protein antigen I / II (Ag I / II), glucosyltransferases (GTFs) and glucan binding proteins (Gbps). Ag I / II, GTFs and GbpB can
provide protection against dental caries. GbpA from S. mutans contains a C-terminal GBD homologous to the GBDs of the GTFs, but the effects of this GBD of GbpA on anticaries immunization has not yet been reported.
METHODS: Balb/c mice were administered with the expressed and purified fusion protein GBD of GbpA. Then the spleen of immunized mice were collected and hybridized with SP2/0 cells. Monoclonal antibodies were prepared with the routine methods. The monoclonal antibodies were identificated by ELISA and Western blot. RESULTS: Six strains of hybridomas were obtained. Titre oi ascites reached dilution of 10-6. Isotype of antibodies were determined. CONCLUSION: Monoclonal antibodies against the GBD of GbpA frorr S.mutans can be obtained by routine hybridoma technic, which will make the sequential preparation of gene technological realized.
第四军医大学硕士学位论文
一
本研究分为两个部分：
一、分泌抗GbPAGBD单克隆抗体细胞系的建立
主要实验内容包括：
1．杂交瘤细胞系的建立：以GBD为抗原，免疫Balb／c ／J’鼠，通过B淋巴
细胞杂交瘤技术，即用B淋巴细胞与骨髓瘤细胞驼／0进行细胞融合，经
筛选和克隆化后，间接ELISA法检测杂交瘤培养上清中有特异性抗体分泌
后，建立杂交瘤细胞系。
2．杂交瘤细胞特性分析：用秋水仙素阻抑法对杂交瘤细胞特性进行染色
体数目分析。
3．效价测定及Ig类别鉴定：用琼脂糖凝胶双向扩散法检测抗体亚类。并
检测腹水效价。
4．单克隆抗体的纯化：用饱和硫酸铰盐析法，对单克隆抗体进行纯化。先
后共融合2次，克隆生长率为100％，阳性率为35％，经过2上次克隆化，
获得了 6株稳定分泌抗GBD的杂交瘤细胞系，分别命名为 2C9jC5J。
3H6、4C9和4FIO，在体外连续培养传代，液氮冻存半年以上，复苏后生
长良好，分泌抗体性能稳定。染色体数目分析结果显示：2C9染色体数为
107．6土3．4条，3C5为 102．2土2．l条，3GI、3H6、4C9和4F10分别为 106．8
i 2．2、102．4 i 3．6、107．ZIZ．7、105．6 i 3．3。说明各细胞系均为融合细胞。
5．ELISA检测抗体效价，六株McAh的腹水效价均为 IX10“‘。2C9、3C5。
3GI和 4C9亚型为 IgGI，。链；3H6和 4F10亚型为 IgGZa，K链。
3
第四军医大学硕士学位论文
二、抗GbpA＊BD单克隆抗体特异性鉴定及相应抗原初步分析
1单克隆抗体的亲和常数：采用间接ELISA法测定MCAb亲和常数。
2 GBD单克隆抗体 Western blot免疫印迹杂交
3免疫组织化学染色
从亲和常数可知 3C5、3H6和 4F10的亲和力大于 2C9、4C9和 3GI的亲
和力。Western blot免疫印迹杂交结果显示，3C5、3H6和 4F10在分子量 43KD
处出现明显的兔疫反应条带，而阴性对照组、空白对照组无阳性条带，2C9。
4C9和3GI在43KD处出现弱阳性条带。至少有3株单抗能够识别变性的
GBD蛋白，证实了单抗的特异性，同时也表明此6株单抗识别空间表位有
所差异。免疫组织化学染色检测结果发现，6株单抗在S，nutans胞壁染色均
呈阳性，进一步证实了其特异性。
Dental caries is one of the three chief non-contagious diseases which threaten human health. Mutans streptococci (MS) are the primary cariogenic agents, and Streptococcus mutans and Streptococcus sobrinus are believed to have the closest relationship with human caries. Adhesion and proliferation of MS are the basis of cariogensis. At present, the study of anticaries mainly focuses on a series of events involving the adherence, accumulation and proliferation of MS on tooth surfaces. Researchers have done many studies on cloning, functions and anticaries immunization of the genes related to adherence and accumulation. These genes includ surface protein antigen I / II (Ag I / II), glucosyltransferases (GTFs) and glucan binding proteins (Gbps). Ag I / II, GTFs and GbpB can
provide protection against dental caries. GbpA from S. mutans contains a C-terminal GBD homologous to the GBDs of the GTFs, but the effects of this GBD of GbpA on anticaries immunization has not yet been reported.
METHODS: Balb/c mice were administered with the expressed and purified fusion protein GBD of GbpA. Then the spleen of immunized mice were collected and hybridized with SP2/0 cells. Monoclonal antibodies were prepared with the routine methods. The monoclonal antibodies were identificated by ELISA and Western blot. RESULTS: Six strains of hybridomas were obtained. Titre oi ascites reached dilution of 10-6. Isotype of antibodies were determined. CONCLUSION: Monoclonal antibodies against the GBD of GbpA frorr S.mutans can be obtained by routine hybridoma technic, which will make the sequential preparation of gene technological realized.
引文
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3. Edwardson S. Characteristics of caries-inducing human streptococci resembling Streptococcus mutans. Arch Oral Biol. 1968; 13: 637-646.
4. Fitzgerald RJ, Keyes PH. Demonstration of the etiologic role of streptococci in experimental caries in hamsters. J Am Dent Asoc. 1960; 61: 9-19.
5. Krasse B. Human streotococci and experimental caries in hamsters. Arch Oral Biol. 1966; 11: 429-436.
6. Loesche WJ, Rowan J, Straffon LH, et al. Association of Streptococcus mutans with human dental decay. Infect Immun. 1975; 11: 1252-1260.
7. Zinner DD, Aran PP, Jablon JM, et al. Expermental caries induced by human streptococci. J Dent Res. 1964; 43: 859-860.
8. Heremans JF, Heremans MT, Schultze HE. Isolation and desceiption of a few properties of the β 2A globulin of human serum, Clin Chim Acta. 1959; 4: 96-102.
9. Tomasi TB, Tan EM, Solomon A. Characteristics of an immune system common to certain external secretions. J Ecp Med. 1965; 21: 101-124.
10. Staat RH, Langdy SD, Doyle RJ. Streptococcus mutans adherence: presumptive evidence for protein-mediated attchment followed by glucan-dependent cellular accumulation. Infect Immun. 1980; 27: 675-681.
11. Marquis RE. Acid-base physiology, stress responses and effects of weak acids on oral streptococci. Abstracts of the 5th national symposium of caries research, society of cariology and endodontology, Chinese stomatological association. Peking, 1998.
12. Michalek SM, Gregory RL, Harmon CC, et al. Protection of gnotobiotic rats against dental caries by passive immunization with bovine milk antibody to Streptococcus mutans. Infect. Immun. 1987; 55: 2340-2347
13．樊明文，陈平，边专。免疫防龋研究的新进展。中华口腔医学杂志。1999，34：69-72
14. Smith DJ, King WF, Godiska R. Passive transfer of immunogobulin Y antibody to Streptococcus mutans glucan binding protein B can confer protection against experimental dental caries. Infect. Immun. 2001; 69: 3135-3142
15. Oho T, Shimazaki Y, Mitoma M, et al. Bovine milk antibodies against cell surface protein antigen PAc-glucosyltransferase fusion protein suppress cell adhesion and alter glucan synthesis of Streptococcus mutans. J Nutri Beth. 1999; 129: 1836-1841
16. Shimazaki Y, Mitoma M, Oho T, et al. Passive immunization with milk produced from an immunized cow prevents oral recolonization by Streptococcus mutans. Clin Diagn Lab Immunol. 2001; 8: 1136-1139
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60. Banas JA, Russell RRB, Ferretti JJ. Sequence analysis of the gene for the glucan-binding protein of Streptococcus mutans Ingbritt. Infect Immun. 1990: 58(3):667-673.
61. Smith DJ, Taubman MA. Experimental immunization of rats with a Streptococcus mutans 59-kilodalton glucan-binding protein protects against dental caries. Infect Immun. 1996; 64(8):3069-3073.
62. Haast W, Banas JA. Ligand-binding properties of the carboxyl-terminal repeat domain of streptococcus mutans glucan-binding protein A. J. Bacteriology 2000; 182:728-733
63．苏凌云、吴补领、李福洋等。变形链球菌葡聚糖结合蛋白A的GBD的分子克隆及在大肠杆菌的表达。牙体牙髓牙周病学杂志，2002：12(4)：185
64. Ma JK, Hiatt M, et al.Generation and assembly ofsecretory antibodies in plants. Science 1995;268:716-719
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66. Ma YM, Lassiter MO,Banas JA, et al. Multiple glucan-binding protein of Streptococcus sobrinus. J Bacterio. 1996; 178:1572-1577
67. Smith DJ, King WF, Wu CD. Structural and antigenic characteristics of Streptococcus sobrinus glucan binding protein. Infect Immun. 1998; 66:5565-5569
2. Carlsson J. A numerical taxonomic study of human oral streptococci. Odontol Revy. 1968; 19: 137-160.
3. Edwardson S. Characteristics of caries-inducing human streptococci resembling Streptococcus mutans. Arch Oral Biol. 1968; 13: 637-646.
4. Fitzgerald RJ, Keyes PH. Demonstration of the etiologic role of streptococci in experimental caries in hamsters. J Am Dent Asoc. 1960; 61: 9-19.
5. Krasse B. Human streotococci and experimental caries in hamsters. Arch Oral Biol. 1966; 11: 429-436.
6. Loesche WJ, Rowan J, Straffon LH, et al. Association of Streptococcus mutans with human dental decay. Infect Immun. 1975; 11: 1252-1260.
7. Zinner DD, Aran PP, Jablon JM, et al. Expermental caries induced by human streptococci. J Dent Res. 1964; 43: 859-860.
8. Heremans JF, Heremans MT, Schultze HE. Isolation and desceiption of a few properties of the β 2A globulin of human serum, Clin Chim Acta. 1959; 4: 96-102.
9. Tomasi TB, Tan EM, Solomon A. Characteristics of an immune system common to certain external secretions. J Ecp Med. 1965; 21: 101-124.
10. Staat RH, Langdy SD, Doyle RJ. Streptococcus mutans adherence: presumptive evidence for protein-mediated attchment followed by glucan-dependent cellular accumulation. Infect Immun. 1980; 27: 675-681.
11. Marquis RE. Acid-base physiology, stress responses and effects of weak acids on oral streptococci. Abstracts of the 5th national symposium of caries research, society of cariology and endodontology, Chinese stomatological association. Peking, 1998.
12. Michalek SM, Gregory RL, Harmon CC, et al. Protection of gnotobiotic rats against dental caries by passive immunization with bovine milk antibody to Streptococcus mutans. Infect. Immun. 1987; 55: 2340-2347
13．樊明文，陈平，边专。免疫防龋研究的新进展。中华口腔医学杂志。1999，34：69-72
14. Smith DJ, King WF, Godiska R. Passive transfer of immunogobulin Y antibody to Streptococcus mutans glucan binding protein B can confer protection against experimental dental caries. Infect. Immun. 2001; 69: 3135-3142
15. Oho T, Shimazaki Y, Mitoma M, et al. Bovine milk antibodies against cell surface protein antigen PAc-glucosyltransferase fusion protein suppress cell adhesion and alter glucan synthesis of Streptococcus mutans. J Nutri Beth. 1999; 129: 1836-1841
16. Shimazaki Y, Mitoma M, Oho T, et al. Passive immunization with milk produced from an immunized cow prevents oral recolonization by Streptococcus mutans. Clin Diagn Lab Immunol. 2001; 8: 1136-1139
17. Munro G, Evans P, Todryk S, et al. A protein fragment of streptococcal cell surface antigen Ⅰ/Ⅱ which prevents adhesion of Streptococcus mutans. Infect. Immun.. 1993; 61: 4590-4598
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