巴氏杆菌toxA基因的原核表达及生物学特性研究
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摘要
为了探讨多杀性巴氏杆菌皮肤坏死毒素(PMT)对真核细胞的致病机制,将编码PMT的tox A基因以及其N端tox N(1~1461 bp)和C端tox C(2 958~3 858 bp)分别克隆到原核表达载体p ET-32a中进行蛋白表达。3个重组蛋白在大肠杆菌中均以可溶性形式表达,其分子质量大小分别为176 ku、88 ku和67 ku,均能与His单克隆抗体发生特异性免疫反应。体外细胞毒性试验和豚鼠皮肤坏死试验均表明,r PMT与天然PMT具有相同的生物学功能。此外,以原核表达蛋白r PMT-N和r PMT-C分别制备的鼠多抗为一抗,对转染表达N端和C端真核质粒的Vero细胞进行IFA和Western-blot检测。结果表明,2种蛋白特异性良好,制备的多克隆抗体免疫反应条带单一。该研究的成功开展将为后续探究PMT细胞内作用机制提供可靠的生物材料。
To investigate PMT of Pasteurella mutocida in pathogenesis of mammal cells,the prokaryotic expression vectors of entire tox A gene,N-terminal(1—1 461 bp) and C-terminal genes(2 958—3 858 bp) were constructed by molecular cloning with p ET-32 a.Three recombinant proteins are expressed and their molecular weighs are 176 ku,88 ku and 67 ku,respectively.In result,the expressed product bands could specifically bind to the anti-HIS-labeled monoclonal antibody.Both the cellular toxicity in vitro test and the dermonecrotic test in guinea pigs showed that the r PMT had equal bioactivities with natural PMT.The anti-r PMT-N and anti-r PMT-C serums were obtained by immunizing ICR mice.PMT-N and PMT-C expressed in Vero cells can be bind to these polyclonal antibodies,indicating that these polyclonal antibodies had high specificity.The aboved-mentioned results provide reliable biomaterials for further studies on PMT mechanisms in eukaryotic cells.
To investigate PMT of Pasteurella mutocida in pathogenesis of mammal cells,the prokaryotic expression vectors of entire tox A gene,N-terminal(1—1 461 bp) and C-terminal genes(2 958—3 858 bp) were constructed by molecular cloning with p ET-32 a.Three recombinant proteins are expressed and their molecular weighs are 176 ku,88 ku and 67 ku,respectively.In result,the expressed product bands could specifically bind to the anti-HIS-labeled monoclonal antibody.Both the cellular toxicity in vitro test and the dermonecrotic test in guinea pigs showed that the r PMT had equal bioactivities with natural PMT.The anti-r PMT-N and anti-r PMT-C serums were obtained by immunizing ICR mice.PMT-N and PMT-C expressed in Vero cells can be bind to these polyclonal antibodies,indicating that these polyclonal antibodies had high specificity.The aboved-mentioned results provide reliable biomaterials for further studies on PMT mechanisms in eukaryotic cells.
引文
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[20]秦峰,刘学良,魏桂林,等.生物医药制剂中内毒素去除研究进展[J].药物生物技术,2003,10(1):53-56.QIN Feng,LIU Xue-liang,WEI Gui-lin,et al.Progress the endotoxin removal from biomedical praparations[J].Pharmaceutical Biotechnology,2003,10(1):53-56.(in Chinese)
[21]MORRISON D C,ULEVITCH R J.The effects of bacterial endotoxins on host mediation systems.A review[J].The American Journal of Pathology,1978,93(2):526-618.
[22]汤细彪,吴斌,赵战勤,等.重组产毒多杀性巴氏杆菌毒素PMT的N-端和C-端蛋白的生物学活性及免疫原性[J].微生物学报,2008,48(2):213-219.TANG Xi-biao,WU Bin,ZHAO Zhan-qin,et al.Characteristics and immunogenicity of the N-teriminal and C-terminal recombinants of Pasteurella multocida toxin[J].Acta Microbiologica Sinca,2008,48(2):213-219.(in Chinese)
[23]胡军勇,汤金梅,汤细彪,等.猪产毒多杀性巴氏杆菌皮肤坏死毒素的细胞毒性及其抗体的制备[J].华中农业大学学报.2010,29(4):479-483.HU Jun-yong,TANG Jin-mei,TANG Xi-biao,et al.Characterization and its antibody preparation of Pasteurella multocida toxin[J].Journal of Huazhong Agricultural University,2010,29(4):479-483.(in Chinese)
[2]FERNANDEZ R M,VACA S,REYESLOPOEZ M,et al.Outer membrane vesicles of Pasteurella multocida contain virulence factors[J].Microbiologyopen,2014,3(5):711-717.
[3]JORDAN R W,HAMILTON T D,HAYES C M,et al.Modulation of the humoral immune response of swine and mice mediated by toxigenic Pasteurella multocida[J].Fems Immunology and Medical Microbiology,2003,39(1):51-59.
[4]PEDERSEN K B,NIELSEN N C.Atrophic Rhinitis in Pigs[M].Luxembourg:Commission of the European Communities,1983.
[5]FOGED N T.Quantitation and purification of the Pasteurella multocida toxin by using monoclonal antibodies[J].Infection and Immunity,1988,56(8):1901-1906.
[6]ELLING F,PEDERSEN K B.The pathogenesis of persistent turbinate atrophy induced by toxigenic Pasteurella multocida in pigs[J].Veterinary Pathology,1985,22(5):469-474.
[7]LAX A J,CHANTER N.Cloning of the toxin gene from Pasteurella multocida and its role in atrophic rhinitis[J].Journal of General and Applied Microbiology,1990,136(1):81-87.
[8]DEJONG M F,OEI H L,TETENBURG G J.AR-pathogenicity Test for Pasteurella multocida Isolates[M].Copenhagen:Proceedings of International Pig Veterinary Society,1980.
[9]PENNINGS A M,STORM P K.A test in vero cell monolayers for toxin production by strains of Pasteurella multocida isolated from pigs suspected of having atrophic rhinitis[J].Veterinary Microbiology,1984,9(5):503-508.
[10]WILSON B A,PONFERRADA V G,VALLANCE J E,et al.Localization of the intracellular activity domain of Pasteurella multocida toxin to the N terminus[J].Infection and Immunity,1999,67(1):80-87.
[11]PULLINGER G D,SOWDHAMINI R,LAX A J.Localization of functional domains of the mitogenic toxin of Pasteurella multocida[J].Infection and Immunity,2001,69(12):7839-7850.
[12]WARD P N,MILES A J,SUMNER I G,et al.Activity of the mitogenic Pasteurella multocida toxin requires an essential C-terminal residue[J].Infection and Immunity,1998,66(12):5636-5642.
[13]FOGED N T,NIELSEN J P,JORSAL S E.Protection against progressive atrophic rhinitis by vaccination with Pasteurella multocida toxin purified by monoclonal antibodies[J].Veterinary Record,1989,125(1):7-11.
[14]彭忠,王庚,吴广敬,等.猪源多杀性巴氏杆菌的分离与鉴定[J].中国兽医科学,2015,45(9):943-951.PENG Zhong,WANG Geng,WU Guang-jing,et al.Isolation and identification of Pasteurella multocida from swine[J].Chinese Veterinary Scinece,2015,45(9):943-951.(in Chinese)
[15]LIU S,TOBIAS R,MCCLURE S,et al.Removal of endotoxin from recombinant protein preparations[J].Clinical Biochemistry,1997,30(6):455-463.
[16]蔡慧丽.生物技术药品分离纯化过程中内毒素的去除[J].海峡药学.2006,18(2):157-159.CAI Hui-li.Removal of endotoxin during the isolation and purification of biotechnolody drugs[J].Strait Pharmaceutical Journal,2006,18(2):157-159.(in Chinese)
[17]WILSON B A,AMINOVA L R,PONFERRADA V G,et al.Differential modulation and subsequent blockade of mitogenic signaling and cell cycle progression by Pasteurella multocida toxin[J].Infection and Immunity,2000,68(8):4531-4538.
[18]PETERSEN S K,FOGED N T.Cloning and expression of the Pasteurella multocida toxin gene,tox A,in Escherichia coli[J].Infection and Immunity,1989,57(12):3907-3913.
[19]王大鹏,吴斌,周锐,等.猪源D型产毒素多杀性巴氏杆菌tox A基因的克隆与表达[J].中国兽医学报,2006,26(3):271-274.WANG Da-peng,WU Bin,ZHOU Rui,et al.Cloning and expression of the tox A gene of Toxigenic Pasteurella multocida serotype D from swine[J].Chinese Journal of Veterinary Science,2006,26(3):271-274.(in Chinese)
[20]秦峰,刘学良,魏桂林,等.生物医药制剂中内毒素去除研究进展[J].药物生物技术,2003,10(1):53-56.QIN Feng,LIU Xue-liang,WEI Gui-lin,et al.Progress the endotoxin removal from biomedical praparations[J].Pharmaceutical Biotechnology,2003,10(1):53-56.(in Chinese)
[21]MORRISON D C,ULEVITCH R J.The effects of bacterial endotoxins on host mediation systems.A review[J].The American Journal of Pathology,1978,93(2):526-618.
[22]汤细彪,吴斌,赵战勤,等.重组产毒多杀性巴氏杆菌毒素PMT的N-端和C-端蛋白的生物学活性及免疫原性[J].微生物学报,2008,48(2):213-219.TANG Xi-biao,WU Bin,ZHAO Zhan-qin,et al.Characteristics and immunogenicity of the N-teriminal and C-terminal recombinants of Pasteurella multocida toxin[J].Acta Microbiologica Sinca,2008,48(2):213-219.(in Chinese)
[23]胡军勇,汤金梅,汤细彪,等.猪产毒多杀性巴氏杆菌皮肤坏死毒素的细胞毒性及其抗体的制备[J].华中农业大学学报.2010,29(4):479-483.HU Jun-yong,TANG Jin-mei,TANG Xi-biao,et al.Characterization and its antibody preparation of Pasteurella multocida toxin[J].Journal of Huazhong Agricultural University,2010,29(4):479-483.(in Chinese)