猪链球菌2型IMPDH基因的克隆表达及相关生物学特性鉴定
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摘要
猪链球菌2型(Streptococcus suis serotype 2，SS_2)是我国猪链球菌病的重要病原，不仅可引起猪脑膜炎、关节炎、心内膜炎、败血症、肺炎和突然死亡，还可感染相关人员并致死，是一种重要的人畜共患病病原。
SS2可区分为强毒株、弱毒株和无毒株，已发现的毒力因子主要包括溶菌酶释放蛋白(MRP)、胞外蛋白因子(EF)、溶血素(SLY)、New ORF2、纤维蛋白原结合蛋白(FBP)等。本实验基于GenBank SS2上述5个主要毒力因子的报道序列，分别在其上下游设计引物，应用不同引物对进行PCR扩增测序，对毒力因子进行基因定位，发现上述基因均位于20kb的片段之中，在这些片段中，发现新的可能毒力因子次黄嘌呤核苷酸脱氢酶(IMPDH)。该基因广泛存在于猪链球菌35个血清型当中，其中大部分为致病型，利用PCR技术将这一毒力因子扩增出目的片段构建了含目的基因的重组表达质粒pET-IMPDH，该质粒转化入宿主菌BL21(DE3)，体外表达蛋白经镍柱亲合层析纯化后活性染色，证实了该蛋白的生物学活性。
1 SS2 IMPDH的发现及生物学特性鉴定
根据SS2的部分测序结果，发现位于已知毒力基因orf2与mrp之间存在两个新的开放阅读框序列。选取可能含有抗原决定簇肽段对应的核酸序列，该阅读框(2738-3694)编码319个氨基酸残基，分子量为33.5kD，与已知任何基因同源性较低。通过InterPro、PHD、DNAstar分析阅读框，并定向克隆至pET-32α(+)载体中，转化至大肠杆菌BL21，表达出分子量为48kD的融合蛋白。该蛋白免疫转印可被SS2的抗血清识别，具有免疫原性；并且含有IMP dehydrogenase结构域，催化IMP生成XMP；纯化蛋白作用于HEp-2细胞，流式细胞仪检测该蛋白可明显延长细胞的S期。
2 SS2 IMPDH重组蛋白的纯化和抗血清的制备
SS2 IMPDH的重组蛋白含有6个连续的组氨酸残基，利用能与Ni~(2+)结合的特性，进行纯化。IMPDH重组蛋白经Ni-NTA His·Bind Resin进行非变性条件
Streptococcus suis serotype 2 is the important pathogen of diseases in pigs and is a major cause of meningitis, septicemia, arthritis, and bronchopneumonia in young pigs and can cause meningitis in humans.
With Streptococcus suis serotype 2, pathogenic, weakly pathogenic, and nonpathogenic strains can be found. The virulent factors that can be found include muraminidase-released protein(MRP), extracellular protein factor(EF), suilysin(SLY), New ORF2, fibronection(Fn). Give the lack of effective vaccines to control Streptococcus suis infection and the lack of a rapid and reliable molecular diagnostic assay to detect its infection, S. suis serotype 2 was sequenced partly in an effort to identify important virulence factors according to five main virulent factors: MRP, EF, SLY, New ORF2 and FBP in GenBank. The gene encoding IMPDH was found. The PCR product of the open reading frame was transformed into E.coli BL21 and the fusion protein of 48 kD was expressed. The recombinant protein was reactive with serum from pigs experimentally infected with virulent strains of S. suis type 2, suggesting that the protein is immunogenic. IMPDH activity straining confirmed that the protein had IMPDH function and can catalyze the rate-limiting reaction of GTP biosynthesis, the NAD-dependent reduction of IMP into XMP. Flow cytometry(FCM) revealed that the protein had apparent effect on HEp-2 cell cycle. 1 Cloning and Expression of Virulence-Associated Determinants of Streptococcus suis Serotype 2
Analysis Gene sequence revealed that a new open reading frame that encoded a polypeptide of 319 amino acid residues with a calculated molecular mass of 33.5 kD was located between orf 2 and mrp of S.suis serotype 2. Second structure was analyzed by InterPro PHD DNAstar software, the PCR product of the open reading frame (2738 — 3694) was transformed into E.coli BL21 and the fusion protein of 48,000MW was expressed. At the amino acid level the deduced primary sequence
SS2可区分为强毒株、弱毒株和无毒株，已发现的毒力因子主要包括溶菌酶释放蛋白(MRP)、胞外蛋白因子(EF)、溶血素(SLY)、New ORF2、纤维蛋白原结合蛋白(FBP)等。本实验基于GenBank SS2上述5个主要毒力因子的报道序列，分别在其上下游设计引物，应用不同引物对进行PCR扩增测序，对毒力因子进行基因定位，发现上述基因均位于20kb的片段之中，在这些片段中，发现新的可能毒力因子次黄嘌呤核苷酸脱氢酶(IMPDH)。该基因广泛存在于猪链球菌35个血清型当中，其中大部分为致病型，利用PCR技术将这一毒力因子扩增出目的片段构建了含目的基因的重组表达质粒pET-IMPDH，该质粒转化入宿主菌BL21(DE3)，体外表达蛋白经镍柱亲合层析纯化后活性染色，证实了该蛋白的生物学活性。
1 SS2 IMPDH的发现及生物学特性鉴定
根据SS2的部分测序结果，发现位于已知毒力基因orf2与mrp之间存在两个新的开放阅读框序列。选取可能含有抗原决定簇肽段对应的核酸序列，该阅读框(2738-3694)编码319个氨基酸残基，分子量为33.5kD，与已知任何基因同源性较低。通过InterPro、PHD、DNAstar分析阅读框，并定向克隆至pET-32α(+)载体中，转化至大肠杆菌BL21，表达出分子量为48kD的融合蛋白。该蛋白免疫转印可被SS2的抗血清识别，具有免疫原性；并且含有IMP dehydrogenase结构域，催化IMP生成XMP；纯化蛋白作用于HEp-2细胞，流式细胞仪检测该蛋白可明显延长细胞的S期。
2 SS2 IMPDH重组蛋白的纯化和抗血清的制备
SS2 IMPDH的重组蛋白含有6个连续的组氨酸残基，利用能与Ni~(2+)结合的特性，进行纯化。IMPDH重组蛋白经Ni-NTA His·Bind Resin进行非变性条件
Streptococcus suis serotype 2 is the important pathogen of diseases in pigs and is a major cause of meningitis, septicemia, arthritis, and bronchopneumonia in young pigs and can cause meningitis in humans.
With Streptococcus suis serotype 2, pathogenic, weakly pathogenic, and nonpathogenic strains can be found. The virulent factors that can be found include muraminidase-released protein(MRP), extracellular protein factor(EF), suilysin(SLY), New ORF2, fibronection(Fn). Give the lack of effective vaccines to control Streptococcus suis infection and the lack of a rapid and reliable molecular diagnostic assay to detect its infection, S. suis serotype 2 was sequenced partly in an effort to identify important virulence factors according to five main virulent factors: MRP, EF, SLY, New ORF2 and FBP in GenBank. The gene encoding IMPDH was found. The PCR product of the open reading frame was transformed into E.coli BL21 and the fusion protein of 48 kD was expressed. The recombinant protein was reactive with serum from pigs experimentally infected with virulent strains of S. suis type 2, suggesting that the protein is immunogenic. IMPDH activity straining confirmed that the protein had IMPDH function and can catalyze the rate-limiting reaction of GTP biosynthesis, the NAD-dependent reduction of IMP into XMP. Flow cytometry(FCM) revealed that the protein had apparent effect on HEp-2 cell cycle. 1 Cloning and Expression of Virulence-Associated Determinants of Streptococcus suis Serotype 2
Analysis Gene sequence revealed that a new open reading frame that encoded a polypeptide of 319 amino acid residues with a calculated molecular mass of 33.5 kD was located between orf 2 and mrp of S.suis serotype 2. Second structure was analyzed by InterPro PHD DNAstar software, the PCR product of the open reading frame (2738 — 3694) was transformed into E.coli BL21 and the fusion protein of 48,000MW was expressed. At the amino acid level the deduced primary sequence
引文
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[1] Staats J J, Feder I, Ok wubua O, et al. Streptococcus suis: Past an present[J]. Vet Res Commun, 1997, 21(6): 381407
[2] 王家政，范明．蛋白质技术手册[M]．北京：科学出版社，2002，21
[3] Ochocka AM, Czyzewska M, Pawelczyk T. Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein. Acta Biochim Pol 2003; 50(1): 239-47
[4] 傅玉才，王进成，张壮志等．细粒棘虫绦虫cDNA克隆EgA31在大肠埃稀氏菌M15中的表达及重组蛋白的纯化和复性．中国兽医科技，2002，32(1)：5—7
[1] Clifton-Hadley, F. A. 1983. Streptococcus suis type 2 infection. Br. Vet. J. 139: 1-5.
[2] Vecht, U., L. A. M. G. van Leengoed, and E. R. M. Verheyen. 1985. Streptococcus suis infection in pigs in the Netherlands (part one). Vet. Q. 7: 315-321
[3] Caparon M G, Stretptococci D S, Ame Oisen, et al. Role of M protein in adherence of group A stretptocci. Micro Pathog, 1992, 12: 199～208
[4] 顾天钊，陆承平，陈怀青．鲍氏不动杆菌MF1株的黏附特性．南京农业大学学报，1999，22(3)：65～68
[5] Jacobs A A C, Loeffen P L W, van den Berg A J G, et al. Identification of a thiol-activated hemolysin of Streptococcus suis. Infect and Immun, 1994, 62: 1742-1748
[6] Gottschalk M G, Lacouture S, Dubreuil J D. Characterization of Streptococcus suis capsular type 2 haemolysin. Microbiol, 1995, 141: 189-195
[7] 曾巧英，陆承平．猪链球菌2型溶菌酶释放蛋白诱导上皮细胞融合和调亡．微生物学报，2003，43(3)：407—412
[8] Weber, G., Olah, E., Lui, M. S., Kizaki, H., Tzeng, D., and Takeda, E. (1980) Adv. Enzyme Regul. 18, 3-26
[9] Collart, F. R., and Huberman, E. (1987) Mol. Cell. Biol. 7, 3328-3331
[10] Gottschalk, M.; Higgins, R. &Bourdreau, M. Use of polyvalent coagglutination reagents for serotyping of Streptococcus suis. J. Clin Microbiol., 31: 2192-2194, 1993
[11] Sala, V.; Antomini, M; Vischi, O. et al. Distribution of capsular types and hemolysin production of Streptococcus suis isolates in northern Italy. International pig veterinary society congress, 14, Bologna, 1996. Bologna, 1996. Proceedings. p. 307
[12] Jacobs, A. A.; Van den Berg, A. J.; Baars, J. C.; Nielsen, B&Johannsen, L.W. Production of suilysin, the thiol-activated haemolysin of Streptococcus suis by field isolates from diseased pigs. Vet. Rec., 137:2 95-296, 1995
2 Chanter N, P W Jones, T J L Alexander. Meningitis in pigs caused by Streptococcus suis-a speculative review. Vet. Microbiol, 1993, 36:39-55
3 Clifton-Hadley F A, T J L Alexander. The carrier site and carrier rate of Streptococcus suis type Ⅱ in pigs. Vet Rec, 1980, 107:40-41
4 Staats J J, Feder I, Ok wu mabua O, et al. Streptococcus suis: Past and present [J]. Vet Res Common, 1997, 21 (6):381-407
5 Staats J J, Feder I, Ok wu mabua O, et al. Streptococcus suis:past and present[J]. Vet Res Commun, 1997, 21 (6):381407.
6 Aarestrup FM, S E Jorsal, N E Jensen. Serological characterization and antimicrobial susceptibility of Streptococcus suis isolates from diagnostic samples in Denmark during 1995 and 1996. Vet Microbiol, 1998, 60:59-66
7 AmassS F, P SanMiguel, L K Clark. Demonstration of vertical transmission of Streptococcus suis in swine by genomic fingerprinting. Clin. Microbiol, 1997, 35:1595-1596
8 Staats J J, LBrandon, B LPlattner, J Nietfeld S Dritz, M MChengappa. Use of ribotyping and hemolysin activity to identify highly virulent Streptococcus suis type 2 isolates. J. Clin. Microbiol, 1998, 36:15-19
9 Quessy S, J D Dubreuil, M Caya, R Higgins. Discrimination of virulent and avirulent Streptococcus suis capsular type 2 isolates from different geographical origins. Infect. Immun, 1995, 63:1975-1979
10 Vecht, U., H. J. Wisselink, Jellema M. L., and Smith H. E.. 1991. Identification of two proteins associated with virulence of Streptococcus suis type 2. Infect. Immun.59:3156-3162
11 Vecht, U., H. J. Wisselink, M. L. Jellema, and H. E. Smith. 1992. Virulence of Streptococcus suis type 2 stains in newborn germfree pigs depends on phenotype. Infect. Immun. 60:550-556
12 Galina, L., U. Vecht, H. J. Wisselink, and C. Pijoan. Prevalence of various phenotypes of Streptococcus suis isolated from swine in the U.S.A. based on the presence of muramidase-released protein and extracellular factor. Can. J. Vet. Res, 1996, 60:72-74
13 Smith H E, U Vecht, H J Wisselink, et al. Mutants of Streptococcus suis types 1 and 2 impaired in expression of muramidase-released protein and extracellular protein induce disease in newborn germfree pigs. Infect Immun, 1996,64:4409-4412
14 Achim Allgaier, Ralph Goethe, Henk J Wisselink. Relatedness of Streptococcus suis Isolates of Various Serotypes and Clinical Backgrounds as Evaluated by Macrorestriction Analysis and Expression of Potential Virulence Traits.Journal of Clinical Microbiology, 2001, 39( 2): 445-453
15 Mogollon J D, C Pijoan, M P Murtaugh, et al. Identification of epidemic strain of Streptococcus suis by genomic fingerprinting. J. Clin. Microbiol, 1991,29:7824-7887
16 Smith H E, M Rijnsburger, N Stockhofe-Zurwieden, et al. Virulent strains of Streptococcus suis serotype 2 and highly virulent strains of Streptococcus suis serotype 1 can be recognized by a unique ribotype profile. J. Clin. Microbiol, 1997,35:1049-1053
17 Achim Allgaier, Ralph Goethe, Henk J Wisselink, et al. Relatedness of Streptococcus suis Isolates of Various Serotypes and Clinical Backgrounds as Evaluated by Macrorestriction Analysis and Expression of Potential Virulence Traits. Journal of Clinical Microbiology, 2001, 39( 2): 445-453
18 Staats J J, B L Plattner, J Nietfeld, et al. Use of ribotyping and hemolysin activity to identify highly virulent Streptococcus suis type 2 isolates, Clin Microbiol, 1998,36:15-19
19 Olsen J E, M Skov. Genomic lineage of Salmonella enterica serovar Dublin. Vet Microbiol, 1984,40:271-282
20 Florence Berthelot-Herault, Corinne Marois, Marcelo Gottschalk, Marylene Kobisch.Genetic Diversity of Streptococcus suis Strains Isolated from Pigs and Humans as Revealed by Pulsed-Field Gel Electrophoresis . Clin Microbio, 2002, 40( 2): 615-619
21 Rasmussen S R, F M Aarestrup, N E Jensen, S E Jorsal. Associations of Streptococcus suis serotype 2 ribotypes profiles with clinical disease and antimicrobial resistance. Clin Microbiol, 1999,37:404-408
22 Ana I Vela, Joaquin Goyache, Carmen Tarradas.Analysis of Genetic Diversity of Streptococcus suis Clinical Isolates from Pigs in Spain by Pulsed-Field Gel Electrophoresis. Clin Microbio, 2003, 41(6): 2498-2502
23 Chatellier S, Gottschalk M, Higgins R, et al. Relatedness of Streptococcus suis serotype 2 isolates from different geographic origins as evaluated by molecular fingerprinting and
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25 Sonia Chatellier, Jose Harel, Ying Zhang, Marcelo Gottschalk, et al.Phylogenetic diversity of Streptococcus suis strains of various serotypes as revealed by 16S rRNA gene sequence comparison. International Journal of Systematic Bacteriology, 1998, 48:581-589
26 Bentley R W, Leigh, J A & Collins, M D. Intra-generic structure of Streptococcus based on comparative analysis of small-subunit rRNA sequences. Int J Syst Bacteriol ,1991,41:487-494.
27 Norton PM, Rolph C, Ward PN, Bentley RW, Leigh JA. Epithelial invasion and cell lysis by virulent strains of Streptococcus suis is enhanced by the presence of suilysin. FEMS Immunol Med Microbiol 1999 Oct;26(1):25-35
28 Lalonde M, Segura M, Lacouture S, Gottschalk M.lnteractions between Streptococcus suis serotype 2 and different epithelial cell lines. Microbiology 2000 Aug;146 ( Pt 8): 1913-21 29Charland N, Nizet V, Rubens CE, Kim KS, Lacouture S, Gottschalk M.Streptococcus suis serotype 2 interactions with human brain microvascular endothelial cells. Infect Immun 2000 Feb;68(2):637-43
30 Haataja S, Tikkanen K, Hytonen J, Finne J. The Gal alpha 1-4 Gal-binding adhesin of Streptococcus suis, a gram-positive meningitis-associated bacterium. Adv Exp Med Biol 1996;408:25-34
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33 Charland N, Harel J, Kobisch M, Lacasse S, Gottschalk M.Streptococcus suis serotype 2 mutants deficient in capsular expression. Microbiology 1998 Feb;144 ( Pt 2):325-32
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35 Segura MA, Cleroux P, Gottschalk M. Streptococcus suis and group B Streptococcus differ in their interactions with murine macrophages. FEMS Immunol Med Microbiol. 1998 Jul;21 (3): 189-95.
36 Tuomanen E.Entry of pathogens into the central nervous system. FEMS Microbiol Rev 1996 Jul;18(4):289-99
37 Smith HE, Damman M et al.Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor. Infect Immun 1999 Apr;67(4): 1750-6
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39 Salasia SI, Lammler C, Herrmann G Properties of a Streptococcus suis isolate of serotype 2 and two capsular mutants. Vet Microbiol 1995 Jul;45(2-3): 151-6
40 Brazeau C, Gottschalk M, Vincelette S, Martineau-Doize B. In vitro phagocytosis and survival of Streptococcus suis capsular type 2 inside murine macrophages. Microbiology 1996 May; 142 (Pt 5): 1231-7
41 Charland N, Harel J, Kobisch M, Lacasse S, Gottschalk M. Streptococcus suis serotype 2 mutants deficient in capsular expression. Microbiology 1998 Feb;144 (Pt 2):325-32
42 Smith HE, Damman M, van der Velde J, Wagenaar F, Wisselink HJ, Stockhofe-Zurwieden N, Smits MA. Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor. Infect Immun 1999 Apr;67(4): 1750-6
43 Mogollon JD, Pijoan C, Murtaugh MP, Cleary PP, Collins JE. Testing meningeal strains of Streptococcus suis to detect M protein genes. Res Vet Sci 1992 Sep;53(2):244-6
44 Smith HE, Vecht U, Gielkens AL, Smits MA. Cloning and nucleotide sequence of the gene encoding the 136-kilodalton surface protein (muramidase-released protein) of Streptococcus suis type 2. Infect Immun 1992 Jun;60(6):2361-7
45 Lalonde M, Segura M, Lacouture S, et al. Interations between Streotococcus suis serotype 2 and different epithelial cell lines. Microbiology, 2000, Aug, 146(8):1913-21
46 Smith HE, Reek FH, Vecht U, Gielkens AL, Smits MA. Repeats in an extracellular protein of weakly pathogenic strains of Streptococcus suis type 2 are absent in pathogenic strains. Infect Immun 1993 Aug;61 (8):3318-26
47 Wisselink HJ, Vecht U et al. Protection of pigs against challenge with virulent Streptococcus suis serotype 2 strains by a muramidase-released protein and extracellular factor vaccine. Vet Rec 2001 Apr 14;148(15):473-7
48 Smith HE, Vecht U, Wisselink HJ, et al. Mutants of Streptococcus suis types 1 and 2 impaired in expression of muramidase-released protein and extracellular protein induce disease in newborn germfree pigs. Infect Immun 1996 Oct;64(10):4409-12
49 Galina L, Vecht U, Wisselink HJ, Pijoan C. Prevalence of various phenotypes of Streptococcus suis isolated from swine in the U.S.A. based on the presence of muraminidase-released protein and extracellular factor. Can J Vet Res 1996 Jan;60(1):72-4
50 I Feder, MM Chengappa, B Fenwick, et al. Partial characterization of Streptococcus suis type 2 hemolysin. J. Clin. Microbiol. 1994 32: 1256-1260.
51 Mariela Segura and Marcelo Gottschalk, Streptococcus suis Interactions with the Murine Macrophage Cell Line J774: Adhesion and Cytotoxicity.Infect. Immun. 2002 70: 4312-4322.
52 Flanagan J, Collin N, Timoney J, Mitchell T, Mumford JA, Chanter N. Characterization of the haemolytic activity of Streptococcus equi. Microb Pathog 1998 Apr;24(4):211-21
53 Norton PM, Rolph C, Ward PN, Bentley RW, Leigh JA. Epithelial invasion and cell lysis by virulent strains of Streptococcus suis is enhanced by the presence of suilysin. FEMS Immunol Med Microbiol 1999 Oct;26(1):25-35
54 Hanski E,Caporan M.Protein F. A fibronectin-binding protein ,is an adhesin of the group A streptococcus pyogenes.Proc Natl Acad Sci USA, 1992,89:6172-6176
55 Astrid de Greeff, Herma Buys, Robin Verhaar, et al.Contribution of Fibronectin-Binding Protein to Pathogenesis of Streptococcus suis Serotype 2Infection and Immunity, 2002,70(3) :1319-1325
56 Chia J S, C Y Yeh, J Y Chen. Identification of a fibronectin binding protein from Streptococcus mutans. Infect Immun, 2000, 68:1864-1870
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58 D Melancon, D Grenier.Production and Properties of Bacteriocin-Like Inhibitory Substances from the Swine Pathogen Streptococcus suis Serotype 2. Applied and Environmental Microbiology, 2003,69( 8): 4482-4488
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[1] Clifton-Hadley, EA. 1983. Streptococcus suis type 2 infection. Br. Vet. J. 139:1-5.
[2] Vecht, U., L. A. M. G. van Leengoed, and E.R.M. Verheyen. 1985. Streptococcus suis infection in pigs in the Netherlands (part one). Vet. Q.7:315-321
[3] Arends, J. P., and H. C. Zanen. 1988. Meningitis caused by Streptococcus suis in humans. Rev. Infect. Dis. 10:131-137
[4] Staats J J, Feder I, Ok wu mabua O, et al. Streptococcus suis: Past and present [J]. Vet Res Common. 1997. 21 (6):381-407
[5] 黄毓茂，黄引贤，余志东，等．2型猪链球菌的初步研究[J]．中国畜禽传染病，1993(5)：1～3
[6] 姚火春，陈国强，陆承平．猪链球菌1998分离株病原特性鉴定[J]．南京农业大学学报，1999，22(2)：67-70
[7] Vecht, U., H. J. Wisselink, Jellema M. L., and Smith H. E.. 1991. Identification of two proteins associated with virulence of Streptococcus suis type 2. Infect. Immun. 59:3156-3162
[8] Vecht, U., H. J. Wisselink, M. L. Jellema, and H. E.Smith. 1992. Virulence of Streptococcus suis type 2 stains in newborn germfree pigs depends on phenotype. Infect. Immun. 60:550-556
[9] Galina L, Vecht, U., Wisselink, H. J., Pijoan C. Prevalence of various phenotypes of Streptococcus suis isolated from swine in the U.S.A. based on the presence of muraminidase-released protein and extracellular factor. Can J Vet Res 1996 Jan; 60(1):72-74
[10] Smith H. E., Herma Buijs, H.. J. Wisselink, et al. Selection of Virulence-Associated determinants of Streptococcus suis serotype 2 by in viro complementation. 2001. Infect. Immun. 69:1961-1966
[11] 李干武，姚火春，陆承平．在猪链球菌2型江苏分离株中发现新的ORF2毒力相关基因．农业生物技术学报，2003，11：295—298
[12] 倪艳秀，何孔旺，王继春。猪链球菌2型的PCR快速检测。中国兽医学报，2002，22(5)：474—476
[13] Jacobs A A. Loeffen P L, VAN den Berg A J, et al. identification, purification, and characterization of a thiol-activated hemolysin(suilysin) of Streptococcus suis [J]. Infect. Immun, 1994, 62(5): 1742-1748
[14] Mary Meehan, Yvoone Lynagh, Caroline Woods, et al. Fibrinogen-bindingprotein(TgBP) of Streptococcus equi subsp, equi additionally binds IgG and contributes to virulence in a mouse model. Microbiology, 2001, 147, 3311-3322
[15] Goldstein, Barry M.; Coly, Thomas D. IMP dehydrogenase: structural aspects of inhibitor binding. Current Medicinal Chemistry (1999), 6(7): 519-536
[16] Colby, Thomas D.; Vanderveen, Kdsten; Strickler, Michael D. et al.Crytal structure of human type Ⅱ inosine monophosphate dehydrogenase: implication for iigand binding and drug design. Proceedings of the National Academy of Sciences of the United States of America(1999), 96(7):3531-3536
[17] Frank R.. Collart ; Eliezer Huberman. Cloning and Sequence Analysis of the human and Chinese Hamster Inosine-5-monophosphate Dehydrogenase cDNAs*. The Journal of Biological Chemistry(1988), 30(263): 15769-15772
[1] Staats J J, Feder I, Ok wubua O, et al. Streptococcus suis: Past an present[J]. Vet Res Commun, 1997, 21(6): 381407
[2] 王家政，范明．蛋白质技术手册[M]．北京：科学出版社，2002，21
[3] Ochocka AM, Czyzewska M, Pawelczyk T. Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein. Acta Biochim Pol 2003; 50(1): 239-47
[4] 傅玉才，王进成，张壮志等．细粒棘虫绦虫cDNA克隆EgA31在大肠埃稀氏菌M15中的表达及重组蛋白的纯化和复性．中国兽医科技，2002，32(1)：5—7
[1] Clifton-Hadley, F. A. 1983. Streptococcus suis type 2 infection. Br. Vet. J. 139: 1-5.
[2] Vecht, U., L. A. M. G. van Leengoed, and E. R. M. Verheyen. 1985. Streptococcus suis infection in pigs in the Netherlands (part one). Vet. Q. 7: 315-321
[3] Caparon M G, Stretptococci D S, Ame Oisen, et al. Role of M protein in adherence of group A stretptocci. Micro Pathog, 1992, 12: 199～208
[4] 顾天钊，陆承平，陈怀青．鲍氏不动杆菌MF1株的黏附特性．南京农业大学学报，1999，22(3)：65～68
[5] Jacobs A A C, Loeffen P L W, van den Berg A J G, et al. Identification of a thiol-activated hemolysin of Streptococcus suis. Infect and Immun, 1994, 62: 1742-1748
[6] Gottschalk M G, Lacouture S, Dubreuil J D. Characterization of Streptococcus suis capsular type 2 haemolysin. Microbiol, 1995, 141: 189-195
[7] 曾巧英，陆承平．猪链球菌2型溶菌酶释放蛋白诱导上皮细胞融合和调亡．微生物学报，2003，43(3)：407—412
[8] Weber, G., Olah, E., Lui, M. S., Kizaki, H., Tzeng, D., and Takeda, E. (1980) Adv. Enzyme Regul. 18, 3-26
[9] Collart, F. R., and Huberman, E. (1987) Mol. Cell. Biol. 7, 3328-3331
[10] Gottschalk, M.; Higgins, R. &Bourdreau, M. Use of polyvalent coagglutination reagents for serotyping of Streptococcus suis. J. Clin Microbiol., 31: 2192-2194, 1993
[11] Sala, V.; Antomini, M; Vischi, O. et al. Distribution of capsular types and hemolysin production of Streptococcus suis isolates in northern Italy. International pig veterinary society congress, 14, Bologna, 1996. Bologna, 1996. Proceedings. p. 307
[12] Jacobs, A. A.; Van den Berg, A. J.; Baars, J. C.; Nielsen, B&Johannsen, L.W. Production of suilysin, the thiol-activated haemolysin of Streptococcus suis by field isolates from diseased pigs. Vet. Rec., 137:2 95-296, 1995