摘要
兴隆水牛是海南著名的地方水牛品种,具有耐湿热、抗病力强等特性,有较高的经济养殖价值。由于缺乏资源保护意识,现有兴隆水牛品种资源退化,抗病力不断下降,这对其生产性能和产品质量产生了较大影响,造成了巨大经济损失。传染疾病的发生己成为影响水牛养殖产业健康发展的主要因素,而对水牛疾病相关蛋白的研究将有助于提高水牛对疾病的综合抗性,并指导其养殖。
Toll样受体(Toll-like receptors, TLR)是天然免疫系统中重要的模式识别受体(Pattern recognition receptors, PRRs),可以识别多种病原相关分子模式(Pathogen-associated molecular patterns, PAMPs),并参与中风、感染、肿瘤和中枢神经系统等疾病相关的破坏性炎症过程。Toll样受体2(Toll-like receptor2, TLR2)是TLR家族重要成员之一,可以调节或控制先天与获得性免疫,识别广泛的PAMPs。其在结核分枝杆菌和革兰氏阳性菌的识别中发挥重要功效,在内源性配体的识别和自身免疫性疾病的发展中也起着重要作用。髓样分化因子88(Myeloid differentiation primary response protein88, MyD88)是TLR信号通路中重要的接头蛋白,也是信号向下游转导的关键靶分子。最近的研究表明MyD88可能在创伤性脑损伤后的炎症和病变发展中起重要作用。基于以上原因,对TLR2和MyD88的研究将有助于指导TLR参与的重病牲畜和人的临床治疗,对TLR信号通路的调节有可能成为炎性免疫性疾病新的治疗策略。本研究通过RT-PCR技术从海南兴隆水牛中分别扩增出TLR2、MyD88cDNA序列,其开放阅读框分别为2355bp和891bp。对TLR2基因进行了生物信息学分析,并对MyD88进行了原核表达,构建了pET28a-MyyD88表达载体,转化E. coli BL21(DE3),经IPTG诱导表达后,进行了Ni柱亲和层析纯化及Western blotting鉴定,得到一个带His-Tag的约39kDa的重组融合蛋白。
细胞分裂周期蛋白42(cell division cycle42, Cdc42)是Rho家族蛋白成员,具有GTP酶活性,在肿瘤细胞转化、增殖、生存、浸润和转移等方面起分子开关作用。其异常活动会导致多种疾病如癌症的发生。最近对Cdc42蛋白的研究主要调查其在病理生理机制中的作用,并评估其在相关疾病中的治疗潜力。本实验克隆了Cdc42cDNA序列,序列分析显示其包含1个576bp的开放阅读框。应用荧光定量PCR和Western blotting技术对Cdc42在兴隆水牛心、肝、脾、肺、肾和小肠六种组织中的表达水平进行了检测,结果显示Cdc42在六种组织中均有表达,但其表达水平具有组织特异性,在肺中的表达量最高。本文所得结果为研究兴隆水牛相关疾病的病理生理机制和提供直接的治疗方法奠定了基础。
Xing long buffalo is a native buffalo breed of Hainan province, which has the characteristics of disease-resistant, economic-value high, and muggy-resistant. But the resources of Xinglong buffalo degenerated seriously for the lack of conservation of the breed resources. The degeneration of the breed influenced the buffalo's performance of production and quality of related product, which brought tremendous economic losses. The cultivation of Xinglong buffalo was threaten by the contagious diseases and reasons above, while the researches of pathological protein of buffalo will help to build up buffaloes'resistance to diseases and direct the future of cultivation.
Toll-like receptors (TLRs) are important Pattern recognition receptors (PRRs) in the innate immune system, which can recognize diversified Pathogen-associated molecular patterns (PAMPs) and have been shown to be involved in the damaging inflammatory processes associated with stroke, infection, neoplasia, and other diseases in the central nervous system. Toll-like receptor2(TLR2) is an important member of TLRs family and can control and adjust the innate and acquired immunity, which has the capability to recognize the widest range of PAMPs. TLR2is important for the recognition of mycobacteria and gram-positive bacteria, and it is an important crossroad between encounter with bacteria and development of self-reactive diseases. Myeloid differentiation primary response protein88(MyD88) is a crucial downstream adaptor protein for TLRs, which is essential for TLRs signaling pathway and pro-inflammatory gene expression. The recent studies have indicated that Myd88may play an important role in inflammation and lesion development after traumatic brain injury. For these reasons, the researches of MyD88and TLRs with the pathophysiologic mechanisms that contribute to organ dysfunction will direct therapeutic approaches to ameliorating such TLR-mediated responses that may potentially be of clinical benefit in critically ill patients and livestock. The cDNA of TLR2and MyD88were cloned with standard PCR method from Xinglong buffalo, which has the open reading frames (ORFs) with2355bp and891bp respectively. After the analysis of bioinformatics, the cDNA of MyDSS was constructed into vectors to give a recombinant expression plasmid. Recombinant pET28a-MyD88fusion proteins with His-tag were expressed as Inclusion body in E.coli BL21(DE3) after induction. We purified the recombinant proteins with the Ni-NTA affinity chromatography, and verified the protein by Western blotting with anti-his antibody.
The cell divisions cycle42(Cdc42), a member of the Rho family of small guanosine triphosphatase (GTPase) proteins, which plays important roles during cell transformation, proliferation, survival, invasion and metastasis of human cancer cells as a molecular switch. The dysfunction of Cdc42is a feature of some diseases including cancer. Recent studies of Cdc42protein have investigated its roles in pathophysiologic mechanisms and assessed its therapeutic potential in related diseases. The cDNA of Cdc42was amplified with576bp by the method above, the transcription level of Cdc42in heart, liver, spleen, lung, kidney, and intestine were then analyzed with western blotting and real time fluorescence quantitative PCR. Cdc42was found to be expressed in all the tissues above, but at different levels. The expression levels of this gene are highest in lung. The results obtained in this paper provided the base of pathophysiologic mechanisms of related diseases for Xinglong buffalo and direct therapeutic approaches in future.
引文
[1]Janeway CA Jr. Approaching the asymptote? Evolution and revolution in immunology[J]. Cold Spring Harb Symp Quant Biol,1989,54:1-13.
[2]Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors [J]. Nat Immunol,2010,11(5):373-384.
[3]Bowie A, O'Neill LA. The interleukin-1 receptor/Toll-like receptor superfamily: signal generators for pro-inflammatory interleukins and microbial products [J]. J Leukoc Biol,2000,67(4):508-514.
[4]Blasius AL, Beutler B. Intracellular toll-like receptors[J]. Immunity,2010,32(3): 305-315.
[5]Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity[J]. Cell, 2006,124(4):783-801.
[6]Galdiero M, Finamore E, Rossano F, et al. Haemophilus influenzae porin induces Toll-like receptor 2-mediated cytokine production in human monocytes and mouse macrophages[J]. Infect Immun,2004,72(2):1204-1209.
[7]Akira S. Innate immunity to pathogens:diversity in receptors for microbial recognition[J]. Immunol Rev,2009,227(1):5-8.
[8]Mancuso G, Midiri A, Biondo C, et al. Bacteroides fragilis-derived lipopolysaccharide produces cell activation and lethal toxicity via toll-like receptor 4[J]. Infect Immun,2005,73(9):5620-5627.
[9]Mancuso G, Gambuzza M, Midiri A, et al. Bacterial recognition by TLR7 in the lysosomes of conventional dendritic cells[J]. Nat Immunol,2009,10(6):587-594.
[10]Piccinini AM, Midwood KS. DAMPening inflammation by modulating TLR signaling[J]. Mediators Inflamm,2010:672395.
[11]Xu Y, Tao X, Shen B, et al. Structural basis for signal transduction by the Toll/interleukin-1 receptor domains[J]. Nature,2000,408(6808):111-115.
[12]刘艳君.广谱模式识别分子Toll-like receptor 2的研究进展[J].免疫学杂志，2002,18(3)：234-236.
[13]Nilsen NJ, Deininger S, Nonstad U, et al. Cellular trafficking of lipoteichoic acid and Toll-like receptor 2 in relation to signaling:role of CD14 and CD36[J]. J Leukoc Biol,2008,84(1):280-291.
[14]Barbalat R, Lau L, Locksley RM, et al. Toll-like receptor 2 on inflammatory monocytes induces type I interferon in response to viral but not bacterial ligands[J]. Nat Immunol,2009,10(11):1200-1207.
[15]Kang JY, Nan X, Jin MS, et al. Recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer[J]. Immunity,2009,31(6):873-884.
[16]Gantner BN, Simmons RM, Canavera SJ, et al. Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2[J], J Exp Med,2003, 197(9):1107-1117.
[17]Gay NJ, Gangloff M. Structure and function of Toll receptors and their ligands[J]. Annu Rev Biochem,2007,76:141-165.
[18]Vogel SN, Fitzgerald KA, Fenton MJ. TLRs:differential adapter utilization by toll-like receptors mediates TLR-specific patterns of gene expression [J]. Mol Interv, 2003,3(8):466-477.
[19]McGettrick AF, O'Neill LA. The expanding family of MyD88-like adaptors in Toll-like receptor signal transduction[J]. Mol Immunol,2004,41(6-7):577-582.
[20]Barton G.M, Kagan JC. A cell biological view of Toll like receptor function: regulation through compartmentalization[J]. Nat Rev Immunol,2009,9(8):535-542.
[21]Hajjar AM, O'Mahony DS, Ozinsky A, et al. Cutting edge:functional interactions between toll-like receptor (TLR) 2 and TLR1 and TLR6 in response to phenol-soluble modulin[J]. J Immunol,2001,166(1):15-19.
[22]Muir A, Soong G, Sokol S, et al. Toll-like receptors in normal and cystic fibrosis airway epithelial cells[J]. Am JRespir Cell Mol Biol,2004,30(6):777-783.
[23]Johnson CM, Lyle EA, Omueti KO, et al. Cutting edge:A common polymorphism impairs cell surface trafficking and functional responses of TLR1 but protects against leprosy. J Immunol,2007,178(12):7520-7524.
[24]Berdeli A, Celik HA, Ozyurek R, et al. TLR-2 gene Arg753Gln polymorphism is strongly associated with acute rheumatic fever in children[J]. J Mol Med,2005, 83(7):535-541.
[25]Chisholm D, Libet L, Hayashi T, et al. Airway peptidoglycan and immunostimulatory DNA exposures have divergent effects on the development of airway allergen hypersensitivities[J]. J Allergy Clin Immunol,2004,113(3):448-454.
[26]Mullick AE, Tobias PS, Curtiss LK. Modulation of atherosclerosis in mice by Toll-like receptor 2[J]. J Clin Invest,2005,115(11):3149-3156.
[27]Kawakami A, Osaka M, Aikawa M, et al. Toll-like receptor 2 mediates apolipoprotein CⅢ-induced monocyte activation[J]. Circ Res,2008,103(12): 1402-1409.
[28]Kormann MS, Depner M, Hartl D, et al. Toll-like receptor heterodimer variants protect from childhood asthma[J]. JAllergy Clin Immunol,2008,122(1):86-92.
[29]Naiki Y, Sorrentino R, Wong MH, et al. TLR/MyD88 and liver X receptor alpha signaling pathways reciprocally control Chlamydia pneumoniae-induced acceleration of atherosclerosis[J]. J Immunol,2008,181(10):7176-7185.
[30]Liu X, Ukai T, Yumoto H, et al. Toll-like receptor 2 plays a critical role in the progression of atherosclerosis that is independent of dietary lipids[J]. Atherosclerosis,2008,196(1):146-154.
[31]Page K, Lierl KM, Hughes VS, et al. TLR2-mediated activation of neutrophils in response to German cockroach frass[J]. J Immunol,2008,180(9):6317-6324.
[32]Phipps S, Lam CE, Kaiko GE, et al. Toll/IL-1 signaling is critical for house dust mite-specific helper T cell type 2 and type 17 responses[J]. Am J Respir Crit Care Med,2009,179(10):883-893.
[33]Goldammer T, Zerbe H, Molenaar A, et al. Mastitis increases mammary mRNA abundance of beta-defensin 5, toll-like-receptor 2 (TLR2), and TLR4 but not TLR9 in cattle[J]. Clin Diagn Lab Immunol,2004,11(1):174-185.
[34]Lord KA, Hoffman-Liebermann B, Liebermann DA. Complexity of the immediate early response of myeloid cells to terminal differentiation and growth arrest includes ICAM-1, Jun-B and histone variants[J]. Oncogene,1990,5(3):387-396.
[35]Lord KA, Hoffman-Liebermann B, Liebermann DA. Nucleotide sequence and expression of a cDNA encoding MyD88, a novel myeloid differentiation primary response gene induced by IL6[J]. Oncogene,1990,5(7):1095-1097.
[36]Hardiman G, Rock FL, Balasubramanian S, et al. Molecular characterization and modular analysis of human MyD88[J]. Oncogene,1996,13(11):2467-2475.
[37]Slack JL, Schooley K, Bonnert TP, et al. Identification of two major sites in the type I interleukin-1 receptor cytoplasmic region responsible for coupling to pro-inflammatory signaling pathways[J]. J Biol Chem,2000,275(7):4670-4678.
[38]Ohnishi H, Tochio H, Kato Z, et al. Structural basis for the multiple interactions of the MyD88 TIR domain in TLR4 signaling[J]. Proc Natl Acad Sci U S A,2009, 106(25):10260-10265.
[39]Feinstein E, Kimchi A, Wallach D, et al. The death domain:a module shared by proteins with diverse cellular functions [J]. Trends Biochem Sci,1995,20(9): 342-344.
[40]方丽娟,孟民杰.髓样分化因子88研究进展[J].广东药学院学报，2011,27(2)：215-217.
[41]Zhu J, Mohan C. Toll-like receptor signaling pathways--therapeutic opportunities [J]. Mediators Inflamm,2010:781235.
[42]Janssens S, Beyaert R. A universal role for MyD88 in TLR/IL-1R-mediated signaling[J]. Trends Biochem Sci,2002,27(9):474-82.
[43]Reiling N, Ehlers S, Holscher C. MyDths and un-TOLLed truths:Sensor, instructive and effector immunity to tuberculosis[J]. Inununol Lett,2008,116(1):15-23.
[44]Yates RM, Russell DG. Phagosome maturation proceeds independently of stimulation of toll-like receptors 2 and 4[J]. Immunity,2005,23(4):409-417.
[45]Ropert C, Franklin BS, Gazzinelli RT. Role of TLRs/MyD88 in host resistance and pathogenesis during protozoan infection:lessons from malaria[J]. Semin Immunopathol,2008,30(1):41-51.
[46]von Bernuth H, Picard C, Jin Z, et al. Pyogenic bacterial infections in humans with MyD88 deficiency [J]. Science,2008,321(5889):691-696.
[47]Bafica A, Scanga CA, Schito M, et al. Influence of coinfecting pathogens on HIV expression:evidence for a role of Toll-like receptors[J]. J Immunol,2004,172(12): 7229-7234.
[48]Baenziger S, Heikenwalder M, Johansen P, et al. Triggering TLR7 in mice induces immune activation and lymphoid system disruption, resembling HIV-mediated pathology[J]. Blood,2009,113(2):377-388.
[49]Weighardt H, Kaiser-Moore S, Vabulas RM, et al. Cutting edge:myeloid differentiation factor 88 deficiency improves resistance against sepsis caused by polymicrobial infection[J]. J Immunol,2002,169(6):2823-2827.
[50]Naiki Y, Michelsen KS, Schroder NW, et al. MyD88 is pivotal for the early inflammatory response and subsequent bacterial clearance and survival in a mouse model of Chlamydia pneumoniae pneumonia[J]. J Biol Chem,2005,280(32): 29242-29249.
[51]Sadanaga A, Nakashima H, Akahoshi M, et al. Protection against autoimmune nephritis in MyD88-deficient MRL/lpr mice[J]. Arthritis Rheum,2007,56(5): 1618-1628.
[52]Su SB, Silver PB, Grajewski RS, et al Essential role of the MyD88 pathway, but nonessential roles of TLRs 2,4, and 9, in the adjuvant effect promoting Thl-mediated autoimmunity[J]. J Immunol,2005,175(10):6303-6310.
[53]Rakoff-Nahoum S, Medzhitov R. Regulation of spontaneous intestinal tumorigenesis through the adaptor protein MyD88[J]. Science,2007,317(5834): 124-127.
[54]徐世军,沈映君.TLR信号途径关键转接分子MyD88的研究进展[J].中结中医药学刊，2007,25(12)：2504-2505.
[55]Loiarro M, Capolunghi F, Fanto N, et al. Pivotal Advance:inhibition of MyD88 dimerization and recruitment of IRAK1 and IRAK4 by a novel peptidomimetic compound[J]. JLeukoc Biol,2007,82(4):801-810.
[56]Bartfai T, Behrens MM, Gaidarova S, et al. A low molecular weight mimic of the Toll/IL-1 receptor/resistance domain inhibits IL-1 receptor-mediated responses [J]. Proc Natl Acad Sci USA,2003,100(13):7971-7976.
[57]Johnson DI, Pringle JR. Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity[J]. J Cell Biol,1990, 111(1):143-152.
[58]Johnson DI. Cdc42:An essential Rho-type GTPase controlling eukaryotic cell polarity[J]. Microbiol Mol Biol Rev,1999,63(1):54-105.
[59]Shinjo K, Koland JG, Hart MJ, et al. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K):identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein Cdc42[J]. Proc Natl Acad Sci USA,1990,87(24):9853-9857.
[60]Wennerberg K, Der CJ. Rho-family GTPases:it's not only Rac and Rho (and I like it)[J]. J Cell Sci,2004,117(8):1301-1312.
[61]Erickson JW, Zhang C, Kahn RA, et al. Mammalian Cdc42 is a brefeldin A-sensitive component of the Golgi apparatus[J]. JBiol Chem,1996,271(43):26850-26854.
[62]Osmani N, Vitale N, Borg JP, et al. Scrib controls Cdc42 localization and activity to promote cell polarization during astrocyte migration. Curr Biol,2006,16(24): 2395-2405.
[63]Stengel K, Zheng Y. Cdc42 in oncogenic transformation, invasion, and tumorigenesis[J]. Cell Signal,2011,23(9):1415-1423.
[64]解英俊.Cdc42-shRNA表达载体的构建及其对肝癌细胞生物学行为的影响[D].吉林大学，2010,17-18.
[65]Heasman SJ, Ridley AJ. Mammalian Rho GTPases:new insights into their functions from in vivo studies[J]. Nat Rev Mol Cell Biol,2008,9(9):690-701.
[66]Yang L, Wang L, Zheng Y. Gene targeting of Cdc42 and Cdc42GAP affirms the critical involvement of Cdc42 in filopodia induction, directed migration, and proliferation in primary mouse embryonic fibroblasts[J]. Mol Biol Cell,2006, 17(11):4675-4685.
[67]Goldstein B, Macara IG. The PAR proteins:fundamental players in animal cell polarization[J]. Dev Cell,2007,13(5):609-622.
[68]Gomes ER, Jani S, Gundersen GG. Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells [J]. Cell, 2005,121(3):451-463.
[69]Chou MM, Masuda-Robens JM, Gupta ML. Cdc42 promotes G1 progression through p70 S6 kinase-mediated induction of cyclin E expression[J]. J Biol Chem, 2003,278(37):35241-35247.
[70]Yasuda S, Oceguera-Yanez F, Kato T, et al. Cdc42 and mDia3 regulate microtubule attachment to kinetochores[J]. Nature,2004,428(6984):767-771.
[71]Wu F, Chen Y, Li Y, et al. RNA-interference-mediated Cdc42 silencing down-regulates phosphorylation of STAT3 and suppresses growth in human bladder-cancer cells[J]. Biotechnol Appl Biochem,2008,49(2):121-128.
[72]王海舟.Cdc42的生物活性[J].萍乡高等专科学校学报，2010,27(3)：78-81.
[73]Mulloy JC, Cancelas JA, Filippi MD, et al. Rho GTPases in hematopoiesis and hemopathies[J]. Blood,2010,115(5):936-947.
[74]Bustelo XR. Understanding Rho/Rac biology in T-cells using animal models[J]. BioEssays,2002,24(7):602-612.
[75]Chuang TH, Hahn KM, Lee JD, et al. The small GTPase Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes[J]. Mol Biol Cell,1997,8(9): 1687-1698.
[76]Na S, Li B, Grewal IS, et al. Expression of activated CDC42 induces T cell apoptosis in thymus and peripheral lymph organs via different pathways [J]. Oncogene,1999,18(56):7966-7974.
[77]Guo F, Hildeman D, Tripathi P, et al. Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis[J]. Proc Natl Acad Sci USA,2010,107(43):18505-18510.
[78]Guo F, Velu CS, Grimes HL, et al. Rho GTPase Cdc42 is essential for B-lymphocyte development and activation[J]. Blood,2009,114(14):2909-2916.
[79]Jaksits S, Bauer W, Kriehuber E, et al. Lipid raft-associated GTPase signaling controls morphology and CD8+ T cell stimulatory capacity of human dendritic cells[J]. J Immunol,2004,173(3):1628-1639.
[80]Garrett WS, Chen LM, Kroschewski R, et al. Developmental control of endocytosis in dendritic cells by Cdc42[J]. Cell,2000,102(3):325-334.
[81]Lammermann T, Renkawitz J, Wu X, et al. Cdc42-dependent leading edge coordination is essential for interstitial dendritic cell migration[J]. Blood,2009, 113(23):5703-5710.
[82]Szczur K, Zheng Y, Filippi MD. The small Rho GTPase Cdc42 regulates neutrophil polarity via CD11b integrin signaling[J]. Blood,2009,114(20):4527-4537.
[83]Szczur K, Xu H, Atkinson S, et al. Rho GTPase CDC42 regulates directionality and random movement via distinct MAPK pathways in neutrophils[J]. Blood,2006, 108(13):4205-4213.
[84]孔玲.RIG-I在toll样受体介导的吞噬过程中的作用[D].中国科学院上海生命科学研究院,2008,22-23.
[85]Kong L, Ge BX. MyD88-independent activation of a novel actin-Cdc42/Rac pathway is required for Toll-like receptor-stimulated phagocytosis [J]. Cell Res,2008, 18(7):745-55.
[86]张党权,谭晓风,杨伟.植物基因cDNA克隆新技术及进展[J].中国生物工程杂志，2002,22(4)：70-74.
[87]宛传丹,黄宇烽,许晓风.人精子蛋白SP22的cDNA克隆、表达及纯化分析[J].中国生物工程杂志，2004,24(12)：59-63.
[88]马华锋,刘杞.大鼠肝再生增强因子编码区的cDNA克隆和原核表达[J].重庆医科大学学报，2002,27(1)：9-11.
[89]王冬蕾,常洪,杨军香,等.8个亚洲水牛群体的遗传结构分析[J].遗传，2007,29(9)：1103-1109.
[90]张毅,孙东晓,俞英,等.家养水牛30个微卫星标记的多重PCR体系建立及其多态性检测[J].遗传，2008,30(1)：59-64.
[91]Ohnishi H, Tochio H, Kato Z, et al. Structural basis for the multiple interactions of the MyD88 TIR domain in TLR4 signaling[J]. Proc Natl Acad Sci U S A,2009, 106(25):10260-10265.
[92]Lin SC, Lo YC, Wu H. Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signaling[J]. Nature,2010,465(7300):885-890.
[93]Loiarro M, Gallo G, Fanto N, et al. Identification of critical residues of the MyD88 death domain involved in the recruitment of downstream kinases[J]. J Biol Chem, 2009,284(41):28093-28103.
[94]Haque N, Bari MS, Hossain MA, et al. An overview of Brucellosis[J]. Mymensingh Med J,2011,20(4):742-747.
[95]Fosgate GT, Diptee MD, Ramnanan A, et al. Brucellosis in domestic water buffalo (Bubalus bubalis) of Trinidad and Tobago with comparative epidemiology to cattle[J]. Trop Anim Health Prod,2011,43(8):1479-1486.
[96]Olsen SC, Stoffregen WS. Essential role of vaccines in brucellosis control and eradication programs for livestock[J]. Expert Rev Vaccines,2005,4(6):915-928.
[97]Guzman-Verri C, Chaves-Olarte E, von Eichel-Streiber C, et al. GTPases of the Rho subfamily are required for Brucella abortus internalization in nonprofessional phagocytes:direct activation of Cdc42[J]. J Biol Chem,2001,276(48): 44435-44443.
[98]Feng JG, Liu Q, Qin X, et al. Clinicopathological pattern and Annexin A2 and Cdc42 status in patients presenting with differentiation and lymphnode metastasis of esophageal squamous cell carcinomas [J]. Mol Biol Rep,2012,39(2):1267-1274.