重组猪胸膜肺炎放线杆菌NLPI蛋白诱导小鼠抗攻击感染的免疫保护力研究
|
摘要
猪传染性胸膜肺炎(porcine contagious pleuropneumonia,PCP)是猪(Sus scrofa)的一种呼吸道疾病,影响世界养猪业,其病原菌为猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP),现有疫苗由于保护效果不甚理想,因此有必要寻找新的疫苗候选分子。本研究检测了猪APP重组NLPI蛋白(recombinant NLPI,rNLPI)的免疫保护作用。根据Gen Bank中已报道的APP nlpi基因序列设计引物对,然后以APP的基因组为模板PCR扩增nlpi基因,测序后进行生物信息学分析,发现该基因(Gen Bank No.:MH027649)由900 bp组成,编码1个含299个氨基酸的蛋白质,为一外膜脂蛋白,此外膜蛋白在多种APP血清型之间具有很高同源性。将nlpi基因亚克隆至p ET32a(+)载体,转化大肠杆菌(Escherichia coli)BL21(DE3),用异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导表达APP rNLPI,并经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDSPAGE)和蛋白质印迹(Western blot,WB)确认。结果表明,在34 k D处有特异性条带,可被相应的阳性血清识别。动物保护实验采用BALB/c小鼠(Mus musculus)进行,酶联免疫吸附法(enzyme-linked immuno sorbent assay,ELISA)用于测定免疫球蛋G(immunoglobulin G,IgG)抗体滴度。结果表明,弗氏不完全佐剂+50μg rNLPI、弗氏不完全佐剂+30μg rNLPI、弗氏不完全佐剂+10μg rNLPI、30μg rNLPI免疫后,可诱导小鼠分别产生40%、20%、10%和20%的存活率;佐剂组和生理盐水对照组存活率为0。幸存小鼠慢慢恢复健康,并正常采食。可见,rNLPI皮下免疫可诱导小鼠产生免疫应答和较高的抗攻击感染的免疫保护力。该研究结果为进一步筛选猪传染性胸膜肺炎分子疫苗积累了基础资料。
Porcine contagious pleuropneumonia(PCP) is a serious respiratory disease with great harm to pigs(Sus scrofa),caused by Actinobacillus pleuropneumoniae(APP) infection.It is one of the major epidemic diseases in the world pig industry.Existing vaccines can not give full protection against App infection,so it is necessary to screening new vaccines.In this study,immune protection of recombinant NLPI(rNLPI)vaccination against App challenged infection was detected.Based on APP nlpi sequences in Gen Bank,the primers were designed.APP nlpi sequence was obtained by PCR from APP genome.Sequence analysis and function predication was made by bioinformatics software.The results revealed that the length of this gene(Gen Bank No.:MH027649) was 900 bp and its predicat protein was a 299 amino acid resides of an puptative outer membrane lipoprotein.Homology analysis showed that it shared high sequence similarity between different serotypes of APP.The APP nlpi was inserted into pET32a(+).Recombinant protein rNLPI was produced in Escherichia coli BL21 after induced by isopropyl β-D-1-thiogalactopyranoside(IPTG) and then determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot.The34 kD-protein was detected and showed good immunogenicity.The protective efficacy of rNLPI experiment was tested in 6-week-old specific-pathogen-free BALB/c mice(Mus musculus).The serum immunoglobulin G(IgG) level was assayed by enzyme-linked immune sorbent assay(ELISA).The results showed that 50 μg rNLPI+Frund's incompleted adjuvant,30 μg rNLPI+ Frund's incompleted adjuvant and 10 μg rNLPI+Frund's incompleted adjuvant and 30 μg rNLPI respectively induced 41%,22%,10% and 20% survival rate.No mice survived the challenge in adjuvant group and control group.The rest living mice of experiment groups slowly returned to normal health status.From above,rNLPI induced a partial immune protection of mice against App challenged infection.The study accumulated data for exploring new vaccines of porcine contagious pleuropneumonia.
Porcine contagious pleuropneumonia(PCP) is a serious respiratory disease with great harm to pigs(Sus scrofa),caused by Actinobacillus pleuropneumoniae(APP) infection.It is one of the major epidemic diseases in the world pig industry.Existing vaccines can not give full protection against App infection,so it is necessary to screening new vaccines.In this study,immune protection of recombinant NLPI(rNLPI)vaccination against App challenged infection was detected.Based on APP nlpi sequences in Gen Bank,the primers were designed.APP nlpi sequence was obtained by PCR from APP genome.Sequence analysis and function predication was made by bioinformatics software.The results revealed that the length of this gene(Gen Bank No.:MH027649) was 900 bp and its predicat protein was a 299 amino acid resides of an puptative outer membrane lipoprotein.Homology analysis showed that it shared high sequence similarity between different serotypes of APP.The APP nlpi was inserted into pET32a(+).Recombinant protein rNLPI was produced in Escherichia coli BL21 after induced by isopropyl β-D-1-thiogalactopyranoside(IPTG) and then determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot.The34 kD-protein was detected and showed good immunogenicity.The protective efficacy of rNLPI experiment was tested in 6-week-old specific-pathogen-free BALB/c mice(Mus musculus).The serum immunoglobulin G(IgG) level was assayed by enzyme-linked immune sorbent assay(ELISA).The results showed that 50 μg rNLPI+Frund's incompleted adjuvant,30 μg rNLPI+ Frund's incompleted adjuvant and 10 μg rNLPI+Frund's incompleted adjuvant and 30 μg rNLPI respectively induced 41%,22%,10% and 20% survival rate.No mice survived the challenge in adjuvant group and control group.The rest living mice of experiment groups slowly returned to normal health status.From above,rNLPI induced a partial immune protection of mice against App challenged infection.The study accumulated data for exploring new vaccines of porcine contagious pleuropneumonia.
引文
杜爱庆,刁有祥,张伟,等.2008.猪胸膜肺炎放线杆菌溶血毒素的克隆表达及其对小鼠免疫保护作用[J].微生物学报,48(3):342-348.(Du A Q,Diao Y X,Zhang W,e al.2008.Cloning and expression of hemolytic-toxin from Actinobacillus pleuropneumoniae and the immunoprotection in mice[J].Acta Microbiologica Sinica,48(3):342-348.)
郝杰,赵墩,刘崇灵,等.2015.猪多杀性巴氏杆菌Plp P基因的原核表达及其免疫保护性的研究[J].中国兽医科学,45(06):584-588.(Hao J,Zhao D,Liu C L,et al.2015.Prokaryotic expression of Plp P gene of pasteurella multocida from swine and study on its immune protective efficacy in mice[J].Chinese Veterinary Science,48(3):342-348.)
韩国全,郭万柱,王利娜等.2009.猪传染性胸膜肺炎新型疫苗的研究进展[J].猪业科学,26(2):25-30.(Han G Q,Guo W Z,Wang L N,et al.2009.Advances on the new vaccines of swine infectious pleuralpneumonia[J].Swine Industry Science,26(2):25-30.)
厚华艳,梁武,吕茂杰,等.2015.猪传染性胸膜肺炎亚单位疫苗研究进展[J].动物医学进展,36(02):84-88.(Hou H Y,Liang W,Lu M J,et al.2015.Advances on subunite vaccines of swine infectious pleuralpneumonia[J].Swine Industry Science,36(02):84-88).
胡佩佩,黄复深,牛俊超,等.2015.猪胸膜肺炎放线杆菌感染小鼠所致炎症中TLR-4信号通路涉及细胞焦亡[J].微生物学报,55(5):650-656.(Hu P P,Huang F S,Niu J C,et al.2015.TLR-4 involvement in pyroptosis of mice with pulmonary inflammation infected by Actinobacillus pleuropneumoniae[J].Acta Microbiologica Sinica,55(5):650-656.)
胡佩佩,杨婷婷,黄复深,等.2017.猪APP r Omp P2诱导小鼠抗攻击感染的免疫保护力研究[J].农业生物技术学报,25(02):299-306.(Hu P P,Yang T T,Huang F S,et al.2017.Immune protection research against challenge infection in mice(Mus musculus)induced by APP r Omp P2 of porcine(Sus scrofa)[J].Journal of Agricultural Biotechnology,25(02):299-306.)
刘燕,庞安娜,韦强,等.2012.兔多杀性巴氏杆菌外膜蛋白A(Omp A)重组蛋白单克隆抗体的制备及潜在应用[J].农业生物技术学报,20(6):676-681.(Liu Y,Pang AN,Wei Q,et al.2012.Preparation and potential application of monoclonal antibody against Pasteurella multocida outer membrane protein A(Omp A)recombinant protein[J].Journal of Agricultural Biotechnology,20(6):676-681.)
雷连成,王磊.2015.猪传染性胸膜肺炎的免疫学致病机制研究进展[J].吉林农业大学学报37(06):631-637.(Lei L C,Wang L.2015.Research progress in immunologic pathogenesis of porcine infectious pleuropneumonia[J].Journal of Jilin Agricultural University,37(06):631-637)
余旭平,邬栋栋,刘晓宁,等.2008.血清1和3型多杀性巴氏杆菌铁调控外膜蛋白共同抗原的初步分析[J].畜牧兽医学报,39(2):245-250.(Yu X P,Wu D D,Liu X N,et al.2015.Primary study on common antigens of iron-regulated outer membrane proteins of serotype 1 and 3 Pasteurella multocida strains[J].Acta Veteri naria et Zoo technica Sin ica,39(2):245-250.)
张飞,曹三杰,文心田,等.2015,猪传染性胸膜肺炎疫苗的研究进展[J].中国兽医学报,35(1 1):1880-1886.(Zhang F,Cao S J,Wen X T,et al.2015.Advance in vaccines against porine contagious pleuropneumonia[J].Chinese Journal of V eterinary Science,35(11):1880-1886.)
张丽芳,罗薇.2014.外膜蛋白的结构、功能及应用研究进展[J].中国畜牧兽医,10:55-61.(Zhang L F,Luo W.2014.Research progress on structure,function and application of outer membrane protein[J].China Animal Husbandry&V eterinary Medicine,10:55-61.)
赵香汝,杨汉春.1997.细菌外膜蛋白的研究现状[J].中国兽医杂志,23(12):41-42.(Zhao X R,Y ang H C.1997.Research progress on bacterial outer membrane proteins[J].Chinese Journal of V eterinary Medicine,23(12):41-42.)
Del Pozo Sacristán R,Michiels A,Martens M,et al.2014.Efficacy of vaccination against Actinobacillus pleuropneumoniae in two belgian farrow-to-finish pig herds with a history of chronic pleurisy[J].V eterinary Record,174(12):302.
Deneer H G,Potter A A.1989.Identification of a maltose-inducible major outer membrane protein in Actinobacillus(Haemophilus)pleuropneumoniae[J].Microbial Pathogenesis,6(6):425-432.
Frey J,Kuhnert P,Villiger L,et al.1996.Cloning and characterization of an Actinobacillus pleuropneumoniae outer membrane protein belonging to the family of PAL lipoproteins[J].Research in microbiology,147(5):351-61.
Gerlach G F,Anderson C,Klashinsky S,et al.1993,Molecular characterization of a protective outer membrane lipoprotein(Om1A)from Actinobacillus pleuropneumoniae serotype 1[J].Infection and immunity,61(2):565-72.
Gonzalez G C,Caamano D L,Schryvers A B.1990.Identification and characterization of a porcine-specific transferrin receptor in Actinobacillus pleuropneumoniae[J].Molecular microbiology,4(7):1173-1179.
Hu X H,Yan H,Liu K,et al.2015.Identification and characterization of a novel stress-responsive outer membrane protein Lip40 from Actinobacillus pleuropneumoniae[J].BMC Biotechnology,15:106.
Schwechheimer C,Kuehn M J.2015.Outer-membrane vesicles from Gram-negative bacteria:Biogenesis and functions[J].Nature Reviews Microbiology,13(10):605-619.
Shao M,Wang Y,Wang C,et al.2010.Evaluation of multicomponent recombinant vaccines against Actinobacillus pleuropneumoniae in mice[J].Acta Veterinaria Scandinavica,52:52-60.
Teng C H,Tseng Y T,Maruvada R,et al.2010.Nlp I contributes to Escherichia coli K1 strain rs218 interaction with human brain microvascular endothelial cells[J].Infection and Immunity,78(7):3090-3096.
Tseng Y T,Wang S W,Kim K S,et al.2012.Nlp I Facilitates deposition of C4bp on Escherichia coli by blocking classical complement-mediated killing,which results in high-level bacteremia[J].Infection and Immunity,80(10):3669-3678.
Zhou Y,Li L,Chen Z H,et al.2013.Adhesion protein Apf Aof Actinobacillus pleuropneumoniae is required for pathogenesis and is a potential target for vaccine development[J].Clinical and Vaccine Immunology,20(2):287-94.
郝杰,赵墩,刘崇灵,等.2015.猪多杀性巴氏杆菌Plp P基因的原核表达及其免疫保护性的研究[J].中国兽医科学,45(06):584-588.(Hao J,Zhao D,Liu C L,et al.2015.Prokaryotic expression of Plp P gene of pasteurella multocida from swine and study on its immune protective efficacy in mice[J].Chinese Veterinary Science,48(3):342-348.)
韩国全,郭万柱,王利娜等.2009.猪传染性胸膜肺炎新型疫苗的研究进展[J].猪业科学,26(2):25-30.(Han G Q,Guo W Z,Wang L N,et al.2009.Advances on the new vaccines of swine infectious pleuralpneumonia[J].Swine Industry Science,26(2):25-30.)
厚华艳,梁武,吕茂杰,等.2015.猪传染性胸膜肺炎亚单位疫苗研究进展[J].动物医学进展,36(02):84-88.(Hou H Y,Liang W,Lu M J,et al.2015.Advances on subunite vaccines of swine infectious pleuralpneumonia[J].Swine Industry Science,36(02):84-88).
胡佩佩,黄复深,牛俊超,等.2015.猪胸膜肺炎放线杆菌感染小鼠所致炎症中TLR-4信号通路涉及细胞焦亡[J].微生物学报,55(5):650-656.(Hu P P,Huang F S,Niu J C,et al.2015.TLR-4 involvement in pyroptosis of mice with pulmonary inflammation infected by Actinobacillus pleuropneumoniae[J].Acta Microbiologica Sinica,55(5):650-656.)
胡佩佩,杨婷婷,黄复深,等.2017.猪APP r Omp P2诱导小鼠抗攻击感染的免疫保护力研究[J].农业生物技术学报,25(02):299-306.(Hu P P,Yang T T,Huang F S,et al.2017.Immune protection research against challenge infection in mice(Mus musculus)induced by APP r Omp P2 of porcine(Sus scrofa)[J].Journal of Agricultural Biotechnology,25(02):299-306.)
刘燕,庞安娜,韦强,等.2012.兔多杀性巴氏杆菌外膜蛋白A(Omp A)重组蛋白单克隆抗体的制备及潜在应用[J].农业生物技术学报,20(6):676-681.(Liu Y,Pang AN,Wei Q,et al.2012.Preparation and potential application of monoclonal antibody against Pasteurella multocida outer membrane protein A(Omp A)recombinant protein[J].Journal of Agricultural Biotechnology,20(6):676-681.)
雷连成,王磊.2015.猪传染性胸膜肺炎的免疫学致病机制研究进展[J].吉林农业大学学报37(06):631-637.(Lei L C,Wang L.2015.Research progress in immunologic pathogenesis of porcine infectious pleuropneumonia[J].Journal of Jilin Agricultural University,37(06):631-637)
余旭平,邬栋栋,刘晓宁,等.2008.血清1和3型多杀性巴氏杆菌铁调控外膜蛋白共同抗原的初步分析[J].畜牧兽医学报,39(2):245-250.(Yu X P,Wu D D,Liu X N,et al.2015.Primary study on common antigens of iron-regulated outer membrane proteins of serotype 1 and 3 Pasteurella multocida strains[J].Acta Veteri naria et Zoo technica Sin ica,39(2):245-250.)
张飞,曹三杰,文心田,等.2015,猪传染性胸膜肺炎疫苗的研究进展[J].中国兽医学报,35(1 1):1880-1886.(Zhang F,Cao S J,Wen X T,et al.2015.Advance in vaccines against porine contagious pleuropneumonia[J].Chinese Journal of V eterinary Science,35(11):1880-1886.)
张丽芳,罗薇.2014.外膜蛋白的结构、功能及应用研究进展[J].中国畜牧兽医,10:55-61.(Zhang L F,Luo W.2014.Research progress on structure,function and application of outer membrane protein[J].China Animal Husbandry&V eterinary Medicine,10:55-61.)
赵香汝,杨汉春.1997.细菌外膜蛋白的研究现状[J].中国兽医杂志,23(12):41-42.(Zhao X R,Y ang H C.1997.Research progress on bacterial outer membrane proteins[J].Chinese Journal of V eterinary Medicine,23(12):41-42.)
Del Pozo Sacristán R,Michiels A,Martens M,et al.2014.Efficacy of vaccination against Actinobacillus pleuropneumoniae in two belgian farrow-to-finish pig herds with a history of chronic pleurisy[J].V eterinary Record,174(12):302.
Deneer H G,Potter A A.1989.Identification of a maltose-inducible major outer membrane protein in Actinobacillus(Haemophilus)pleuropneumoniae[J].Microbial Pathogenesis,6(6):425-432.
Frey J,Kuhnert P,Villiger L,et al.1996.Cloning and characterization of an Actinobacillus pleuropneumoniae outer membrane protein belonging to the family of PAL lipoproteins[J].Research in microbiology,147(5):351-61.
Gerlach G F,Anderson C,Klashinsky S,et al.1993,Molecular characterization of a protective outer membrane lipoprotein(Om1A)from Actinobacillus pleuropneumoniae serotype 1[J].Infection and immunity,61(2):565-72.
Gonzalez G C,Caamano D L,Schryvers A B.1990.Identification and characterization of a porcine-specific transferrin receptor in Actinobacillus pleuropneumoniae[J].Molecular microbiology,4(7):1173-1179.
Hu X H,Yan H,Liu K,et al.2015.Identification and characterization of a novel stress-responsive outer membrane protein Lip40 from Actinobacillus pleuropneumoniae[J].BMC Biotechnology,15:106.
Schwechheimer C,Kuehn M J.2015.Outer-membrane vesicles from Gram-negative bacteria:Biogenesis and functions[J].Nature Reviews Microbiology,13(10):605-619.
Shao M,Wang Y,Wang C,et al.2010.Evaluation of multicomponent recombinant vaccines against Actinobacillus pleuropneumoniae in mice[J].Acta Veterinaria Scandinavica,52:52-60.
Teng C H,Tseng Y T,Maruvada R,et al.2010.Nlp I contributes to Escherichia coli K1 strain rs218 interaction with human brain microvascular endothelial cells[J].Infection and Immunity,78(7):3090-3096.
Tseng Y T,Wang S W,Kim K S,et al.2012.Nlp I Facilitates deposition of C4bp on Escherichia coli by blocking classical complement-mediated killing,which results in high-level bacteremia[J].Infection and Immunity,80(10):3669-3678.
Zhou Y,Li L,Chen Z H,et al.2013.Adhesion protein Apf Aof Actinobacillus pleuropneumoniae is required for pathogenesis and is a potential target for vaccine development[J].Clinical and Vaccine Immunology,20(2):287-94.