猪肺炎支原体P97基因抗原决定簇R1区重组表达质粒的构建、表达及活性鉴定
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摘要
猪肺炎支原体是引起猪气喘病的主要病原。黏附因子是支原体致病的关键因子。本研究根据已报道的序列为参考，设计一对带有EcoR Ⅰ和Hind Ⅲ酶切位点的引物，并通过引物的定点突变，利用PCR方法从猪肺炎支原体168F485株扩增到其黏附因子P97基因的抗原决定簇R1区。扩增产物经双酶切后，重组到表达载体pET-32a(+)中。重组质粒经酶切鉴定，PCR鉴定和测序，结果证实猪肺炎支原体黏附因子P97基因的抗原决定簇R1区定向插入了质粒pET-32a(+)，且阅读框架正确。将其转化宿主菌BL21(DE3)的感受态细胞，然后经过诱导条件(诱导温度、诱导剂用量和诱导时间)的优化，结果发现在37℃下，IPTG终浓度为0.2mmol／L，诱导4h进行表达，可得到了一分子量约265kDa的融合蛋白，表达量可达约23.3％。通过SDS-PAGE分离的表达产物和有机溶剂提纯的表达产物分别经Western Blot和ELISA检测证实具有抗原反应原性，从而为猪气喘病免疫学检测试剂盒和基因工程疫苗的研制奠定了基础。
Mycoplasma hyopneumoniae (Mhp) is the etiologic agent of mycoplasma pneumonia in swine. Attachment to host tissue is essential for colonization by most mucosal pathogens, such as Mycoplasma hyopneumoniae. According to the reported sequence of P97 gene in strain 168, a pair of primers was designed and the Rl region of P97 gene in strain 168F485 was amplicated by PCR. In this case, two restriction sites(EcoR I , Hind III) were added to the primers (P1,P2) separately. The PCR product was inserted into expression plasmid pET-32a(+) after restriction digest. Then the recombinant plasmid was identified by endonuclease analysis, PCR ampliation and DNA sequencing. The report showed that the recombinant plasmid had right open reading frame. It was transformed into competent cells of BL21(DE3), then was induced to express by 0.2mmol/L IPTG at
37℃ for 4 hours .The recombinant protein was nearly 23.3% of the total products. It was proved that the recombinant protein had immune activity through Western blot and ELISA analysis. This provided the basis for the future preparation of Mycoplasma hyopneumoniae test kit and the new vaccine against Mycoplasma hyopneumoniae.
Mycoplasma hyopneumoniae (Mhp) is the etiologic agent of mycoplasma pneumonia in swine. Attachment to host tissue is essential for colonization by most mucosal pathogens, such as Mycoplasma hyopneumoniae. According to the reported sequence of P97 gene in strain 168, a pair of primers was designed and the Rl region of P97 gene in strain 168F485 was amplicated by PCR. In this case, two restriction sites(EcoR I , Hind III) were added to the primers (P1,P2) separately. The PCR product was inserted into expression plasmid pET-32a(+) after restriction digest. Then the recombinant plasmid was identified by endonuclease analysis, PCR ampliation and DNA sequencing. The report showed that the recombinant plasmid had right open reading frame. It was transformed into competent cells of BL21(DE3), then was induced to express by 0.2mmol/L IPTG at
37℃ for 4 hours .The recombinant protein was nearly 23.3% of the total products. It was proved that the recombinant protein had immune activity through Western blot and ELISA analysis. This provided the basis for the future preparation of Mycoplasma hyopneumoniae test kit and the new vaccine against Mycoplasma hyopneumoniae.
引文
1. Leman AD, T Stein B Straw and HD Hilley. Pneumoniae of Swine:its cost and value of control. Mod Vet Pract. 1982,63:195～198
2. Thacker EL, Halbur PG, Ross RF, et al. Mycoplasma hyopneumoniae potentiation of Porcine reproductive and respiratory syndrome virus-induced pneumoniae. J Clin Microbiol, 1999,37(3):620～627
3．金洪效，毛洪先编著．猪气喘病的诊断与防制．江苏科学技术出版社．1993
4. Muirhead MR. Respiratory disease of pigs. Br Vet J. 1979,135:45～52
5. RF Ross. Mycoplasma disease. Disease of Swine 6th edit Iowa State University Press. 1986
6. Whittiestone P. Enzootic pneumoniae of pigs (EPP). Adv Vet Sci Comp Med. 1973,17:1～55
7．蔡宝祥．家畜传染病学．北京：中国农业出版社．1996
8．毕丁仁，王桂枝．动物霉形体及研究方法．北京：中国农业出版社．1998，9
9．彭万强．猪气喘病免疫的探讨．农业出版社．1987，7
10．甘肃农业大学主编．兽医微生物学(第二版)．农业出版社．1994
11. Geary SJ, Walczak EM. Cytopathic effect of whole cells and purified membranes of Mycoplasma hyopneumoniae. Infect Immun. 1983, 41(1):132～136
12. Geary SJ, Walczak EM. Isolation of a cytopathic factor from Mycoplasma hyopneumoniae. Infect Immun. 1985,48(2):576～578
13．张映，耿丹，聂向庭．猪肺炎霉形体膜制备方法对膜活性的研究．中国兽医科技．2002，32(3)：21～22
14．刘桂林，聂向庭，张映．猪肺炎霉形体及其膜致细胞病变的研究．畜牧与兽医．1994，26(4)：149～150
15．陈嘉棣．猪气喘病原霉形体膜致乳兔肾细胞病变作用．畜牧兽医学报．1981，12(3)：181
16. Tajima, M. etal. interaction of Mycoplasma hyopneumonia with porcine respiratory epithelium as observed by election microscopy. Infect Immun, 1982,37:1162～1169
17．张启敬等．猪肺炎霉形体兔化弱毒株的超微结构及致病性的关系．电子显微学报，1989，8(4)：13～18
18．张启敬，等．猪肺炎霉形体兔化弱毒株的超微结构及致病性的关系．电子显微学报，1990，9(1)：5～9．
19. Mebus. C.A., N. R. Underdahl. Scanning electron microscopy of trachea and bronchi from gnotobiotic pigs inoculated with Mycoplasma hyopneumonia.
Am.J. Vet. Res. 1977,38:1249～1254
20．张启敬等．猪肺炎霉行体乳兔继代株的超微结构及与宿主细胞的相互作用．中国兽药监察所研究汇集．1985～1986
21. DeBey, M.C. & Ross, R.F. Ciliostasis and loss of cilia induced by Mycoplasma hyopneumoniae in porcine tracheal organ cultures. Infect Immun. 1994,62:5312
22. Razin, S. P.,Jacobs, E. Mycoplasma adhesin. J. Gen. Microbiol. 1992,138:407
23. Zhang Q, Young TF, Ross RF. Glycolipid receptors for attachment of Mycoplasma hyopneumoniae to porcine respiratory ciliated cells. Infect Immun 1994, 62(10):4367～4373
24. Beachey, R. H. Bacterial adherence: adhesion-receptor interactions mediating the attaehement of bacteria to mucosal surfaces. J. Infect. Dis. 1981,143: 325～345
25. Collier, A. M. Attachment by mycoplasmas and its role in disease. Rev. Infect. Dis. 1983,5:s685～s691
26. Blanchard B, Vena MM, Cavalier A, Le Lannic J, Gouranton J, Kobisch M. Electron microscopic observation of the respiratory tract of SPF piglets inoculated with Mycoplasma hyopneumoniae. Vet Microbiol 1992,30(4): 329～341
27. Zhang Q, Young TF, Ross RF. Identification and characterization of a Mycoplasma hyopneumoniae adhesin. Infect Immun. 1995,63(3):1013～1019
28. Hsu, T., and F. C. Minion. Molecular analysis of the P97 cilium adhesin operon of Mycoplasma hyopneumoniae. Gene 1998,214:13～23
29. Hsu T., S. Artiushin, and F. C. Minion. Cloning and analysis of P97, a respiratory cilium adhesin gene of Mycoplasma hyopneumoniae. J. Bacteriol. 1997,179:1317～1323
30. Wilton JL, Scarman AL, Walker MJ, Djordjevic SP. Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae. Microbiology. 1998, 144 (7):1931～1943
31. Williamson, M. P, The structure and function of proline-rich regions in proteins. Biochem J. 1994,297,249～261
32. Jody L. Wilton, Anthony L. Scarman, Mark J. Walker etal. Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae. Microbiology. 1998,144:1931～1943
33. Hsu, T., and F. C. Minion. Identification of the cilium binding epitope of the Mycoplasma hyopneumoniae P97 adhesin. Infect. Immun. 1998, 66:4762～4766
34. F.C. Minion., Adams. C, Hsu T. Rl region of P97 mediates adherence of Mycoplasma hyopneumoniae to swine cilia. Infect Immun. 2000,68(5): 3056～3060
35. Young, T.F. Chiang, Y. K Ross, R.F. Evaluation of local and systemic humoral immune response to Mycoplasma hyopneumoniae. Proceedings of the 11th Congress of the International Pig Veterinary Society. 1990,9
36. Muto A, S Osawa. The guanine and cytosine content of genomic DNA and bacterial evolution. Proc Natl Acad Sci. 1987, 84:166～169
37. Tully JG and S Razin. Molecular and diagnostic procedures in Mycoplasmology vol Ⅱ. Diagnostic Procedure Academic Press, 1996.
38. Himmelreich R, H Hilbert, H Plagens, et al. Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acid Res, 1996, 24: 4420
39. Notarnicola SM, McIntosh MA, Wise KS. Multiple translational products from a Mycoplasma hyorhinis gene expressed in Escherichia coli. J Bacteriol. 1990, 172(6) :2986～2995.
40. Futo S, Seto Y, Mitsuse S, Mori Y, Suzuki T. Kawai K. Molecular cloning of a 46-kilodalton surface antigen (P46) gene from Mycoplasma hyopneumoniae: direct evidence of CGG codon usage for arginine. J Bacteriol. 1995, 177(7) : 1915～1917
41. Yamao F, Muto Y, Kawauchi et al. UGA is read as tryptophan in Mycoplasma capricolum. Proc. Natl. Acad. Sci. 1985, 82:2306～2309
42．刘茂车，张映．霉形体检测技术简介．中国兽医科技．2002，32(3)：41～43
43. IDEXX. Mycoplasma hyopneumoniae Antibody fest Kit. USA
44. Shimoji Y, Oishi E, Muneta Y, etal. Vaccine efficacy of the attenuated Erysipelotbrix rhusiopathiae YS-19 expressing a recombinant protein of Mycoplasma hyopneumoniae P97 adhesin against mycoplasmal pneumonia of swine. Vaccine 2003, 21 (5-6) :532～537
45．曹玉璞，叶元康主编．支原体与支原体病(第一版)．北京：人民卫生出版社．2000
46．贾广乐．猪肺炎支原体P97基冈的PCR扩增、序列分析及原核表达 山西农业大学硕士论文．2002，5
47．于海东．猪肺炎支原体P97基因的PCR扩增、序列分析．河北农业大学硕士论文．2000，5
48. Joseph Sam(?)ok, David W. Russell. Molecular Cloning: A Laboratory Manual,3rd ed. Cold Spring Harbor Laboratory Press. 2001
49．奥斯伯(Ausubel,F.)(美)等著；颜子颖，王海林译．精编分子生物学实验指南．科学出版社．1998，6
50．蒋鹤春．伪狂犬病病毒gE基因的表达及初步应用．南京农业大学硕士论文．2001，5
51．(?)新安，刘秀梵．实验室手册．江苏农学院传染病教研组．1990，6
52. (?)awa S, TH Jukes, K Watannabe and A Muto. Recent. evidence for evolution of the genetic code. Microbiol Rev.1992,56;229
53. Grosjean H, Fiers W. Preferential condon usage in prokaryotic genes: the optimal condon anticondon interaction energy and the selective condon usage in efficiently expressed genes. Gene. 1982,18:199～209
54．静国忠．基因工程及其分子生物学基础．北京大学出版社．1999，8
55．pET System Manual．Novagen
56．王玮蓁，孔繁荣，朱学骏等．蛋白表达在支原体研究中的应用．中国皮肤性病学杂志．1999，13(4)：195～197
57. LaVallie ER, OiBlasio EA, Kovacic S, et al. h thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology. 1993,11(2):187～193
58．顾觉奋主编．分离纯化工艺原理．北京：中国医药科技出版社．1996，10
59．陈浩，陈于红，朱德煦等．重组蛋白质纯化技术．中国生物工程杂志．2002，22(5)：87～92
60. Panagiotidis CA, Silver sterstein SJ. Pales a dual tag prokaryotic expression vector for the purification of full length proteins. Gene. 1995,164(1): 45～47
2. Thacker EL, Halbur PG, Ross RF, et al. Mycoplasma hyopneumoniae potentiation of Porcine reproductive and respiratory syndrome virus-induced pneumoniae. J Clin Microbiol, 1999,37(3):620～627
3．金洪效，毛洪先编著．猪气喘病的诊断与防制．江苏科学技术出版社．1993
4. Muirhead MR. Respiratory disease of pigs. Br Vet J. 1979,135:45～52
5. RF Ross. Mycoplasma disease. Disease of Swine 6th edit Iowa State University Press. 1986
6. Whittiestone P. Enzootic pneumoniae of pigs (EPP). Adv Vet Sci Comp Med. 1973,17:1～55
7．蔡宝祥．家畜传染病学．北京：中国农业出版社．1996
8．毕丁仁，王桂枝．动物霉形体及研究方法．北京：中国农业出版社．1998，9
9．彭万强．猪气喘病免疫的探讨．农业出版社．1987，7
10．甘肃农业大学主编．兽医微生物学(第二版)．农业出版社．1994
11. Geary SJ, Walczak EM. Cytopathic effect of whole cells and purified membranes of Mycoplasma hyopneumoniae. Infect Immun. 1983, 41(1):132～136
12. Geary SJ, Walczak EM. Isolation of a cytopathic factor from Mycoplasma hyopneumoniae. Infect Immun. 1985,48(2):576～578
13．张映，耿丹，聂向庭．猪肺炎霉形体膜制备方法对膜活性的研究．中国兽医科技．2002，32(3)：21～22
14．刘桂林，聂向庭，张映．猪肺炎霉形体及其膜致细胞病变的研究．畜牧与兽医．1994，26(4)：149～150
15．陈嘉棣．猪气喘病原霉形体膜致乳兔肾细胞病变作用．畜牧兽医学报．1981，12(3)：181
16. Tajima, M. etal. interaction of Mycoplasma hyopneumonia with porcine respiratory epithelium as observed by election microscopy. Infect Immun, 1982,37:1162～1169
17．张启敬等．猪肺炎霉形体兔化弱毒株的超微结构及致病性的关系．电子显微学报，1989，8(4)：13～18
18．张启敬，等．猪肺炎霉形体兔化弱毒株的超微结构及致病性的关系．电子显微学报，1990，9(1)：5～9．
19. Mebus. C.A., N. R. Underdahl. Scanning electron microscopy of trachea and bronchi from gnotobiotic pigs inoculated with Mycoplasma hyopneumonia.
Am.J. Vet. Res. 1977,38:1249～1254
20．张启敬等．猪肺炎霉行体乳兔继代株的超微结构及与宿主细胞的相互作用．中国兽药监察所研究汇集．1985～1986
21. DeBey, M.C. & Ross, R.F. Ciliostasis and loss of cilia induced by Mycoplasma hyopneumoniae in porcine tracheal organ cultures. Infect Immun. 1994,62:5312
22. Razin, S. P.,Jacobs, E. Mycoplasma adhesin. J. Gen. Microbiol. 1992,138:407
23. Zhang Q, Young TF, Ross RF. Glycolipid receptors for attachment of Mycoplasma hyopneumoniae to porcine respiratory ciliated cells. Infect Immun 1994, 62(10):4367～4373
24. Beachey, R. H. Bacterial adherence: adhesion-receptor interactions mediating the attaehement of bacteria to mucosal surfaces. J. Infect. Dis. 1981,143: 325～345
25. Collier, A. M. Attachment by mycoplasmas and its role in disease. Rev. Infect. Dis. 1983,5:s685～s691
26. Blanchard B, Vena MM, Cavalier A, Le Lannic J, Gouranton J, Kobisch M. Electron microscopic observation of the respiratory tract of SPF piglets inoculated with Mycoplasma hyopneumoniae. Vet Microbiol 1992,30(4): 329～341
27. Zhang Q, Young TF, Ross RF. Identification and characterization of a Mycoplasma hyopneumoniae adhesin. Infect Immun. 1995,63(3):1013～1019
28. Hsu, T., and F. C. Minion. Molecular analysis of the P97 cilium adhesin operon of Mycoplasma hyopneumoniae. Gene 1998,214:13～23
29. Hsu T., S. Artiushin, and F. C. Minion. Cloning and analysis of P97, a respiratory cilium adhesin gene of Mycoplasma hyopneumoniae. J. Bacteriol. 1997,179:1317～1323
30. Wilton JL, Scarman AL, Walker MJ, Djordjevic SP. Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae. Microbiology. 1998, 144 (7):1931～1943
31. Williamson, M. P, The structure and function of proline-rich regions in proteins. Biochem J. 1994,297,249～261
32. Jody L. Wilton, Anthony L. Scarman, Mark J. Walker etal. Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae. Microbiology. 1998,144:1931～1943
33. Hsu, T., and F. C. Minion. Identification of the cilium binding epitope of the Mycoplasma hyopneumoniae P97 adhesin. Infect. Immun. 1998, 66:4762～4766
34. F.C. Minion., Adams. C, Hsu T. Rl region of P97 mediates adherence of Mycoplasma hyopneumoniae to swine cilia. Infect Immun. 2000,68(5): 3056～3060
35. Young, T.F. Chiang, Y. K Ross, R.F. Evaluation of local and systemic humoral immune response to Mycoplasma hyopneumoniae. Proceedings of the 11th Congress of the International Pig Veterinary Society. 1990,9
36. Muto A, S Osawa. The guanine and cytosine content of genomic DNA and bacterial evolution. Proc Natl Acad Sci. 1987, 84:166～169
37. Tully JG and S Razin. Molecular and diagnostic procedures in Mycoplasmology vol Ⅱ. Diagnostic Procedure Academic Press, 1996.
38. Himmelreich R, H Hilbert, H Plagens, et al. Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acid Res, 1996, 24: 4420
39. Notarnicola SM, McIntosh MA, Wise KS. Multiple translational products from a Mycoplasma hyorhinis gene expressed in Escherichia coli. J Bacteriol. 1990, 172(6) :2986～2995.
40. Futo S, Seto Y, Mitsuse S, Mori Y, Suzuki T. Kawai K. Molecular cloning of a 46-kilodalton surface antigen (P46) gene from Mycoplasma hyopneumoniae: direct evidence of CGG codon usage for arginine. J Bacteriol. 1995, 177(7) : 1915～1917
41. Yamao F, Muto Y, Kawauchi et al. UGA is read as tryptophan in Mycoplasma capricolum. Proc. Natl. Acad. Sci. 1985, 82:2306～2309
42．刘茂车，张映．霉形体检测技术简介．中国兽医科技．2002，32(3)：41～43
43. IDEXX. Mycoplasma hyopneumoniae Antibody fest Kit. USA
44. Shimoji Y, Oishi E, Muneta Y, etal. Vaccine efficacy of the attenuated Erysipelotbrix rhusiopathiae YS-19 expressing a recombinant protein of Mycoplasma hyopneumoniae P97 adhesin against mycoplasmal pneumonia of swine. Vaccine 2003, 21 (5-6) :532～537
45．曹玉璞，叶元康主编．支原体与支原体病(第一版)．北京：人民卫生出版社．2000
46．贾广乐．猪肺炎支原体P97基冈的PCR扩增、序列分析及原核表达 山西农业大学硕士论文．2002，5
47．于海东．猪肺炎支原体P97基因的PCR扩增、序列分析．河北农业大学硕士论文．2000，5
48. Joseph Sam(?)ok, David W. Russell. Molecular Cloning: A Laboratory Manual,3rd ed. Cold Spring Harbor Laboratory Press. 2001
49．奥斯伯(Ausubel,F.)(美)等著；颜子颖，王海林译．精编分子生物学实验指南．科学出版社．1998，6
50．蒋鹤春．伪狂犬病病毒gE基因的表达及初步应用．南京农业大学硕士论文．2001，5
51．(?)新安，刘秀梵．实验室手册．江苏农学院传染病教研组．1990，6
52. (?)awa S, TH Jukes, K Watannabe and A Muto. Recent. evidence for evolution of the genetic code. Microbiol Rev.1992,56;229
53. Grosjean H, Fiers W. Preferential condon usage in prokaryotic genes: the optimal condon anticondon interaction energy and the selective condon usage in efficiently expressed genes. Gene. 1982,18:199～209
54．静国忠．基因工程及其分子生物学基础．北京大学出版社．1999，8
55．pET System Manual．Novagen
56．王玮蓁，孔繁荣，朱学骏等．蛋白表达在支原体研究中的应用．中国皮肤性病学杂志．1999，13(4)：195～197
57. LaVallie ER, OiBlasio EA, Kovacic S, et al. h thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology. 1993,11(2):187～193
58．顾觉奋主编．分离纯化工艺原理．北京：中国医药科技出版社．1996，10
59．陈浩，陈于红，朱德煦等．重组蛋白质纯化技术．中国生物工程杂志．2002，22(5)：87～92
60. Panagiotidis CA, Silver sterstein SJ. Pales a dual tag prokaryotic expression vector for the purification of full length proteins. Gene. 1995,164(1): 45～47