鲍曼不动杆菌外膜蛋白A慢病毒载体的构建及其对RAW264.7细胞NLRP3炎症小体的激活
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摘要
目的建立OmpA过表达慢病毒载体并观察其对RAW264.7细胞NLRP3炎症小体的激活。方法以鲍曼不动杆菌ATCC 19606为模板,设计引物扩增OmpA基因,将其克隆至慢病毒表达载体pCDH-MSCV-copGFP中。将此表达质粒和辅助质粒共转染HEK293T细胞包装病毒,浓缩病毒上清液并用GFP报告基因法测定病毒滴度;用包装病毒颗粒感染RAW264.7细胞48h,Real-time PCR法检测OmpA的mRNA表达水平,用Western blot法检测OmpA蛋白以及感染后NLRP3炎症小体相关蛋白。结果成功构建OmpA过表达慢病毒载体并在HEK293T细胞中包装得到病毒,经测定病毒滴毒为1.5×109 TU/mL;重组OmpA病毒感染RAW264.7细胞表达OmpA蛋白,该蛋白能引起NLRP3炎症小体激活。结论成功构建OmpA过表达慢病毒载体并证明OmpA可激活RAW264.7细胞NLRP3炎症小体。
Objectives To construct a lentivirus vector for outer membrane protein A(OmpA)of Acinetobacter baumannii and to investigate its effect on activation of the NLRP3 inflammasome in RAW264.7 cells. Methods With A.baumannii ATCC 19606 as a template,the OmpA gene was amplified using PCR and inserted into the lentiviral expression vector pCDH-MSCV-copGFP.The expression plasmid and auxiliary plasmid were co-transfected into HEK293 Tcells to package lentiviral virus particles.Afterwards,the supernatant fluid was concentrated,and the viral titer was determined with a GFP reporter gene assay.Virus particles carrying OmpA were used to infect RAW264.7 cells for 48 hours.The level of OmpA mRNA expression in RAW264.7 cells was detected using real-time PCR.The levels of OmpA and NLRP3 inflammasome-related protein expression were detected using Western blotting.An enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of IL-1βin culture supernatant. Results Restriction enzyme digestion and sequencing analysis verified that the OmpA lentivirus vector was successfully constructed,and sufficient lentiviruses were obtained from packaging cells.The viral titer was determined to be 1.5 x109 TU/mL.The expression of OmpA mRNA in RAW264.7 cells infected with the OmpA lentivirus increased significantly after 48 hours,and the level was 2,900 times higher than that in control group.Up-regulated OmpA expression significantly activates NLRP3 inflammasome-related proteins in RAW264.7 cells.In addition,the level of Caspase-1 p10 expression was higher and the release of IL-1βin cell culture supernatant was greater than the level of expression and the amount of release in the empty plasmid group and the control group. Conclusion The recombinant vector pCDH-MSCV-copGFP--OmpA was successfully constructed for overexpression of the OmpA gene,and results demonstrated that OmpA activates the NLRP3 inflammasome in RAW264.7 cells.
Objectives To construct a lentivirus vector for outer membrane protein A(OmpA)of Acinetobacter baumannii and to investigate its effect on activation of the NLRP3 inflammasome in RAW264.7 cells. Methods With A.baumannii ATCC 19606 as a template,the OmpA gene was amplified using PCR and inserted into the lentiviral expression vector pCDH-MSCV-copGFP.The expression plasmid and auxiliary plasmid were co-transfected into HEK293 Tcells to package lentiviral virus particles.Afterwards,the supernatant fluid was concentrated,and the viral titer was determined with a GFP reporter gene assay.Virus particles carrying OmpA were used to infect RAW264.7 cells for 48 hours.The level of OmpA mRNA expression in RAW264.7 cells was detected using real-time PCR.The levels of OmpA and NLRP3 inflammasome-related protein expression were detected using Western blotting.An enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of IL-1βin culture supernatant. Results Restriction enzyme digestion and sequencing analysis verified that the OmpA lentivirus vector was successfully constructed,and sufficient lentiviruses were obtained from packaging cells.The viral titer was determined to be 1.5 x109 TU/mL.The expression of OmpA mRNA in RAW264.7 cells infected with the OmpA lentivirus increased significantly after 48 hours,and the level was 2,900 times higher than that in control group.Up-regulated OmpA expression significantly activates NLRP3 inflammasome-related proteins in RAW264.7 cells.In addition,the level of Caspase-1 p10 expression was higher and the release of IL-1βin cell culture supernatant was greater than the level of expression and the amount of release in the empty plasmid group and the control group. Conclusion The recombinant vector pCDH-MSCV-copGFP--OmpA was successfully constructed for overexpression of the OmpA gene,and results demonstrated that OmpA activates the NLRP3 inflammasome in RAW264.7 cells.
引文
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[16]Munoz-Planillo R,Kuffa P,Martinez-Colon G,et al.K+efflux is the common trigger of NLRP3inflammasome activation by bacterial toxins and particulate matter[J].Immunity,2013,38(6):1142-53.
[2]Dettori M,Piana A,Deriu MG,et al.Outbreak of multidrug-resistant Acinetobacter baumannii in an intensive care unit[J].New Microbiol,2014,37(2):185-91.
[3]Choi CH,Lee JS,Lee YC,et al.Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells[J].BMC Microbiol,2008(8):216.
[4]Jin JS,Kwon SO,Moon DC,et al.Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles[J].PLoS One,2011(6):e17027.
[5]Choi CH,Hyun SH,Lee JY,et al.Acinetobacter baumannii outer membrane protein A targets the nucleus and induces cytotoxicity[J].Cell Microbiol.2008(10):309-19.
[6]Yamaguchi Y,Kurita-Ochiai T,Kobayashi R,et al.Regulation of the NLRP3inflammasome in Porphyromonas gingivalis-accelerated periodontal disease[J].Inflamm Res,2017,66(1):59-65.
[7]Ceballos-Olvera I,Sahoo M,Miller MA,et al.Inflammasomedependent pyroptosis and IL-18 protect against Burkholderia pseudomallei lung infection while IL-1βis deleterious[J].PLoSPathog,2011(7):e1002452.
[8]Kang MJ,Jo SG,Kim DJ,et al.NLRP3inflammasome mediates interleukin-1βproduction in immune cells in response to Acinetobacter baumannii and contributes to pulmonary inflammation in mice[J].Immunology,2017,150(4):495-505.
[9]Dikshit N,Kale SD,Khameneh HJ,et al.NLRP3inflammasome pathway has a critical role in the host immunity against clinically relevant Acinetobacter baumannii pulmonary infection[J].Mucosal Immunol,2018,11(1):257-72.
[10]Borsotti C,Borroni E,Follenzi A.Lentiviral vector interactions with the host cell[J].Curr Opin Virol.2016(21):102-8.
[11]Tokgun O,Fiorentino FP,Tokgun PE,et al.Design of a lentiviral vector for the inducible expression of myc:a new strategy for construction approach[J].Mol Biotechnol,2017,59(6):200-6.
[12]Latz E,Xiao TS,Stutz A,et al.Activation and regulation of the inflammasomes[J].Nat Rev Immunol,2013,13(6):397.
[13]He Y,Hara H,Nunez G.Mechanism and regulation of NLRP3inflammasome activation[J].Trends Biochem Sci,2016,41(12):1012-21.
[14]Kim JJ Jo EK.NLRP3Inflammasome and host protection against bacterial infection[J].J Korean Med Sci,2013,28(10):1415-23.
[15]张涵,索绪斌,张云凌,等.芩百清肺浓缩丸对肺炎支原体感染BALB/c小鼠NLRP3炎性体的影响[J].中国病原生物学杂志,2017,12(10):943-6.
[16]Munoz-Planillo R,Kuffa P,Martinez-Colon G,et al.K+efflux is the common trigger of NLRP3inflammasome activation by bacterial toxins and particulate matter[J].Immunity,2013,38(6):1142-53.