类鼻疽杆菌Ⅲ型分泌系统BPSS1617蛋白的重组表达及其抗体制备
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摘要
目的构建重组表达类鼻疽杆菌Ⅲ型分泌系统蛋白BPSS1617,并制备其抗体。方法采用PCR扩增BPSS1617全长序列,克隆至原核表达载体p ET-22b,将重组表达质粒转化至大肠杆菌BL21(DE3)中进行诱导表达,免疫新西兰兔,制备其多克隆抗体,并通过Western blot、免疫荧光和ELISA检测抗体的特异性,并分析该蛋白在类鼻疽杆菌中的亚细胞定位。结果成功扩增类鼻疽杆菌BPC006株基因组中的BPSS1617基因;酶切和测序验证重组表达质粒p ET-22b-BPSS1617构建成功;经IPTG诱导表达、包涵体洗涤、纯化以及变性复性,成功获得纯度>95%,分子量为25×103的目的蛋白;将纯化的BPSS1617重组蛋白免疫新西兰兔,获得抗BPSS1617蛋白多克隆抗体,经实验验证其特异性良好;同时BPSS1617蛋白主要定位于类鼻疽杆菌的细胞质中。结论成功构建、表达、纯化获得BPSS1617蛋白,并成功制备了其特异性多克隆抗体,并且该蛋白定位于类鼻疽杆菌的细胞质中。
Objective To construct a recombinant expression system for BPSS1617,a component of the type Ⅲ protein secretion system of Burkhlderia pseudomallei,and prepare polyclonal antibodies for this protein. Methods The full-length coding sequence of BPSS1617 was amplified with PCR and cloned into the prokaryotic expression plasmid p ET-22 b. The recombinant plasmid was transformed into E. coli BL21(DE3)strain for expression of the target protein,which,after purification,was used to immunize New Zealand white rabbits to obtain the polyclonal antibodies of BPSS1617. The specificity of the antibodies was evaluated by Western blotting, immunofluorescence assay and enzyme-linked immunosorbent assay(ELISA). The subcellular localization of BPSS1617 in Burkhlderia pseudomallei cells was investigated using the obtained antibodies. Results The results of enzyme digestion and sequencing analysis verified the successful construction of the recombinant p ET-22 b-BPSS1617 expression plasmid. After IPTG induction of E. coli,inclusion body cleansing,purification,solubilization and renaturation,we obtained the target protein(relative molecular mass of 25 × 103) with a purity over 95%. From the white rabbits immunized with this protein,we obtained highly specific polyclonal antibodies against BPSS1617. Using these antibodies,we identified that BPSS1617 protein was mainly localized in the cytoplasm of Burkhlderia pseudomallei cells. Conclusion We successfully obtain the recombinant BPSS1617 protein,and its specific polyclonal antibodies and identify that BPSS1617 is localized mainly in the cytoplasm of Burkhlderia pseudomallei cells.
Objective To construct a recombinant expression system for BPSS1617,a component of the type Ⅲ protein secretion system of Burkhlderia pseudomallei,and prepare polyclonal antibodies for this protein. Methods The full-length coding sequence of BPSS1617 was amplified with PCR and cloned into the prokaryotic expression plasmid p ET-22 b. The recombinant plasmid was transformed into E. coli BL21(DE3)strain for expression of the target protein,which,after purification,was used to immunize New Zealand white rabbits to obtain the polyclonal antibodies of BPSS1617. The specificity of the antibodies was evaluated by Western blotting, immunofluorescence assay and enzyme-linked immunosorbent assay(ELISA). The subcellular localization of BPSS1617 in Burkhlderia pseudomallei cells was investigated using the obtained antibodies. Results The results of enzyme digestion and sequencing analysis verified the successful construction of the recombinant p ET-22 b-BPSS1617 expression plasmid. After IPTG induction of E. coli,inclusion body cleansing,purification,solubilization and renaturation,we obtained the target protein(relative molecular mass of 25 × 103) with a purity over 95%. From the white rabbits immunized with this protein,we obtained highly specific polyclonal antibodies against BPSS1617. Using these antibodies,we identified that BPSS1617 protein was mainly localized in the cytoplasm of Burkhlderia pseudomallei cells. Conclusion We successfully obtain the recombinant BPSS1617 protein,and its specific polyclonal antibodies and identify that BPSS1617 is localized mainly in the cytoplasm of Burkhlderia pseudomallei cells.
引文
[1]LIMMATHUROTSAKUL D,GOLDING N,DANCE D A,et al.Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis[J].Nat Microbiol,2016,1:15008.DOI:10.1038/nmicrobiol.2015.8.
[2]WIERSINGA W J,VAN DER POLL T,WHITE N J,et al.Melioidosis:insights into the pathogenicity of Burkholderia pseudomallei[J].Nat Rev Microbiol,2006,4(4):272-282.DOI:10.1038/nrmicro1385.
[3]毛旭虎.加强类鼻疽的研究[J].第三军医大学学报,2011,33(13):1315-1317.MAO X H.Progress on melioidosis[J].J Third Mil Med Univ,2011,33(13):1315-1317.
[4]吴华,王旭明,黄东良.海南类鼻疽的流行病学特点和临床特点调查研究[J].中国全科医学,2013,16(8):923-926.DOI:10.3969/j.issn.1007-9572.2013.08.027.WU H,WANG X M,HUANG D L.Epidemiological and clinical features of melioidosis in Hainan[J].Chin Gen Prac,2013,16(8):923-926.DOI:10.3969/j.issn.1007-9572.2013.08.027.
[5]KUNDANGAR R S,BHAT S N,MOHANTY S P.Melioidosis mimicking tubercular cold abscess[J].BMJ Case Rep,2017,2017.pii:bcr-2017-221787.DOI:10.1136/bcr-2017-221787.
[6]FANG Y,CHEN H,LI Y L,et al.Melioidosis in Hainan,China:a retrospective study[J].Trans R Soc Trop Med Hyg,2015,109(10):636-642.DOI:10.1093/trstmh/trv065.
[7]VANDER BROEK C W,STEVENS J M.TypeⅢsecretion in the melioidosis pathogen Burkholderia pseudomallei[J].Front Cell Infect Microbiol,2017,7:255.DOI:10.3389/fcimb.2017.00255.
[8]胡艺,胡志强,马腾飞,等.类鼻疽杆菌Ⅲ型分泌系统BPSS1395蛋白表达及其抗体制备与鉴定[J].第三军医大学学报,2017,39(10):941-945.DOI:10.16016/j.1000-5404.201611152.HU Y,HU Z Q,MA T F,et al.Expression and identification of recombinant Burkholderia pseudomallei typeⅢsecretion system BPSS1395 protein and preparation of its polyclonal antibodies[J].J Third Mil Med Univ,2017,39(10):941-945.DOI:10.16016/j.1000-5404.201611152.
[9]CURRIE B J.Melioidosis:evolving concepts in epidemiology,pathogenesis,and treatment[J].Semin Respir Crit Care Med,2015,36(1):111-125.DOI:10.1055/s-0034-1398389.
[10]HEMARAJATA P,BAGHDADI J D,HOFFMAN R,et al.Burkholderia pseudomallei:challenges for the clinical microbiology laboratory[J].J Clin Microbiol,2016,54(12):2866-2873.DOI:10.1128/JCM.01636-16.
[11]STONE J K,DESHAZER D,BRETT P J,et al.Melioidosis:molecular aspects of pathogenesis[J].Expert Rev Anti Infect Ther,2014,12(12):1487-1499.DOI:10.1586/14787210.2014.970634.
[12]JOOMPA P,PONNIKORN S,ROYTRAKUL S,et al.Sittiruk roytrakul,3 investigation of host-pathogen interaction between Burkholderia pseudomallei and autophagy-related protein LC3 using hydrophobic chromatography-based technique[J].Cell Biosci,2017,7(1):45.DOI:10.1186/s13578-017-0172-4.
[13]KANG W T,VELLASAMY K M,RAJAMANI L,et al.Burkholderia pseudomallei typeⅢsecreted protein Bip C:role in actin modulation and translocation activities required for the bacterial intracellular lifecycle[J].Peer J,2016,4(12):e2532.DOI:10.7717/peerj.2532.
[14]VANDER BROEK C W,ZAINAL ABIDIN N,STEVENS J M.Bip C,a predicted Burkholderia pseudomallei type 3 secretion system translocator protein with actin binding activity[J].Front Cell Infect Microbiol,2017,7:333.DOI:10.3389/fcimb.2017.00333.
[15]DU J,REEVES A Z,KLEIN J A,et al.The typeⅢsecretion system apparatus determines the intracellular niche of bacterial pathogens[J].Proc Natl Acad Sci USA,2016,113(17):4794-4799.DOI:10.1073/pnas.1520699113.
[16]Stevens M P,Wood M W,Taylor L A,et al.An inv/axispa-like typeⅢprotein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen[J].Mol Microbiol,2002,46(3):649-659.DOI:10.1046/j.1365-2958.2002.03190.x.
[17]WINSTANLEY C,HALES B A,HART C A.Evidence for the presence in Burkholderia pseudomallei of a typeⅢsecretion system-associated gene cluster[J].J Med Microbiol,1999,48(7):649-656.DOI:10.1099/00222615-48-7-649.
[18]LEE Y H,CHEN Y,OUYANG X,et al.Identification of tomato plant as a novel host model for Burkholderia pseudomallei[J].BMC Microbiol,2010,10:28.DOI:10.1186/1471-2180-10-28.
[2]WIERSINGA W J,VAN DER POLL T,WHITE N J,et al.Melioidosis:insights into the pathogenicity of Burkholderia pseudomallei[J].Nat Rev Microbiol,2006,4(4):272-282.DOI:10.1038/nrmicro1385.
[3]毛旭虎.加强类鼻疽的研究[J].第三军医大学学报,2011,33(13):1315-1317.MAO X H.Progress on melioidosis[J].J Third Mil Med Univ,2011,33(13):1315-1317.
[4]吴华,王旭明,黄东良.海南类鼻疽的流行病学特点和临床特点调查研究[J].中国全科医学,2013,16(8):923-926.DOI:10.3969/j.issn.1007-9572.2013.08.027.WU H,WANG X M,HUANG D L.Epidemiological and clinical features of melioidosis in Hainan[J].Chin Gen Prac,2013,16(8):923-926.DOI:10.3969/j.issn.1007-9572.2013.08.027.
[5]KUNDANGAR R S,BHAT S N,MOHANTY S P.Melioidosis mimicking tubercular cold abscess[J].BMJ Case Rep,2017,2017.pii:bcr-2017-221787.DOI:10.1136/bcr-2017-221787.
[6]FANG Y,CHEN H,LI Y L,et al.Melioidosis in Hainan,China:a retrospective study[J].Trans R Soc Trop Med Hyg,2015,109(10):636-642.DOI:10.1093/trstmh/trv065.
[7]VANDER BROEK C W,STEVENS J M.TypeⅢsecretion in the melioidosis pathogen Burkholderia pseudomallei[J].Front Cell Infect Microbiol,2017,7:255.DOI:10.3389/fcimb.2017.00255.
[8]胡艺,胡志强,马腾飞,等.类鼻疽杆菌Ⅲ型分泌系统BPSS1395蛋白表达及其抗体制备与鉴定[J].第三军医大学学报,2017,39(10):941-945.DOI:10.16016/j.1000-5404.201611152.HU Y,HU Z Q,MA T F,et al.Expression and identification of recombinant Burkholderia pseudomallei typeⅢsecretion system BPSS1395 protein and preparation of its polyclonal antibodies[J].J Third Mil Med Univ,2017,39(10):941-945.DOI:10.16016/j.1000-5404.201611152.
[9]CURRIE B J.Melioidosis:evolving concepts in epidemiology,pathogenesis,and treatment[J].Semin Respir Crit Care Med,2015,36(1):111-125.DOI:10.1055/s-0034-1398389.
[10]HEMARAJATA P,BAGHDADI J D,HOFFMAN R,et al.Burkholderia pseudomallei:challenges for the clinical microbiology laboratory[J].J Clin Microbiol,2016,54(12):2866-2873.DOI:10.1128/JCM.01636-16.
[11]STONE J K,DESHAZER D,BRETT P J,et al.Melioidosis:molecular aspects of pathogenesis[J].Expert Rev Anti Infect Ther,2014,12(12):1487-1499.DOI:10.1586/14787210.2014.970634.
[12]JOOMPA P,PONNIKORN S,ROYTRAKUL S,et al.Sittiruk roytrakul,3 investigation of host-pathogen interaction between Burkholderia pseudomallei and autophagy-related protein LC3 using hydrophobic chromatography-based technique[J].Cell Biosci,2017,7(1):45.DOI:10.1186/s13578-017-0172-4.
[13]KANG W T,VELLASAMY K M,RAJAMANI L,et al.Burkholderia pseudomallei typeⅢsecreted protein Bip C:role in actin modulation and translocation activities required for the bacterial intracellular lifecycle[J].Peer J,2016,4(12):e2532.DOI:10.7717/peerj.2532.
[14]VANDER BROEK C W,ZAINAL ABIDIN N,STEVENS J M.Bip C,a predicted Burkholderia pseudomallei type 3 secretion system translocator protein with actin binding activity[J].Front Cell Infect Microbiol,2017,7:333.DOI:10.3389/fcimb.2017.00333.
[15]DU J,REEVES A Z,KLEIN J A,et al.The typeⅢsecretion system apparatus determines the intracellular niche of bacterial pathogens[J].Proc Natl Acad Sci USA,2016,113(17):4794-4799.DOI:10.1073/pnas.1520699113.
[16]Stevens M P,Wood M W,Taylor L A,et al.An inv/axispa-like typeⅢprotein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen[J].Mol Microbiol,2002,46(3):649-659.DOI:10.1046/j.1365-2958.2002.03190.x.
[17]WINSTANLEY C,HALES B A,HART C A.Evidence for the presence in Burkholderia pseudomallei of a typeⅢsecretion system-associated gene cluster[J].J Med Microbiol,1999,48(7):649-656.DOI:10.1099/00222615-48-7-649.
[18]LEE Y H,CHEN Y,OUYANG X,et al.Identification of tomato plant as a novel host model for Burkholderia pseudomallei[J].BMC Microbiol,2010,10:28.DOI:10.1186/1471-2180-10-28.
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