猪肺炎支原体纤毛黏附因子P97和F7-CTB对O型口蹄疫灭活疫苗的免疫增强作用
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摘要
旨在研究猪肺炎支原体(Mycoplasma hyopneumoniae,简称Mhp)纤毛黏附因子P97以及F7鞭毛蛋白(flagellin,简称F)-霍乱毒素B亚基(cholera toxin B subunit,简称CTB)2种重组蛋白对口蹄疫(foot-and-mouth disease,简称FMD)灭活疫苗的免疫增强作用。通过体外表达获得pCold-F7-CTB、pET32a-P97-R1 2种重组蛋白,用间接酶联免疫吸附试验(enzyme-linked immunosorbent assay,简称ELISA)方法检测pCold-F7-CTB蛋白的生物学活性,将重组蛋白分别与口蹄疫病毒(foot-and-mouth disease virus,简称FMDV)灭活抗原、口蹄疫灭活疫苗混合制备,皮下或腹腔接种Balb/c小鼠,共免疫2次,间隔3周。分别于免疫前、免疫后21、35、49 d采血,49 d后无菌摘取脾脏。采用阻断ELISA的方法检测小鼠血清口蹄疫O型病毒免疫球蛋白(IgG)抗体滴度,用流式细胞术检测脾淋巴细胞亚型比例,以评估免疫效力。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,简称SDS-PAGE)和蛋白质印迹法(Western Blot)分析结果表明,2种重组蛋白成功表达。用GMl(神经节苷脂)-ELISA方法验证pCold-F7-CTB的生物学活性,发现融合蛋白F7-CTB保留了与GMl的结合能力。小鼠免疫试验结果表明,经皮下注射接种后,单独口蹄疫病毒灭活抗原组没有产生明显的抗体水平,而F7-CTB免疫增强剂组比单独口蹄疫病毒灭活抗原组的IgG滴度更高。在试验中发现,当重组蛋白与口蹄疫灭活疫苗联合使用时,血清中的IgG滴度明显增高,CD3~+CD8~+T淋巴细胞比值上升;P97和F7-CTB混合使用对口蹄疫灭活疫苗的免疫增强效果最佳。可以初步得出,免疫增强剂P97和F7-CTB具有协同作用,皮下注射可显著提高FMD的特异性IgG水平,CD3~+CD8~+T淋巴细胞比值升高,显示其具有良好的应用前景。
引文
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[23]韦艳娜,熊祺琰,董璐,等.猪肺炎支原体P97R1抗原免疫刺激复合物的制备及对活疫苗免疫刺激能力的增强作用研究[J].中国预防兽医学报,2013,35(12):1016-1019.
[24]Batah J,Denève-Larrazet C,Jolivot P A,et al.Clostridium difficile flagella predominantly activate TLR5-linked NF-kappa B pathway in epithelial cells[J].Anaerobe,2016,38:116-124.
[25]Tahoun A,Jensen K,Corripio-Miyar Y,et al.Functional analysis of bovine TLR5 and association with IgA responses of cattle following systemic immunisation with H7 flagella[J].Veterinary Research,2015,46:9.
[26]Stratmann T.Cholera toxin subunit B as adjuvant-an accelerator in protective immunity and a break in autoimmunity[J].Vaccines,2015,3(3):579-596.
[27]孙小涵.非致病性大肠杆菌鞭毛蛋白增强FMDV免疫原性的研究[D].长春:吉林农业大学,2017.徐磊,孙博怿,盛鹏程,等.湖州地区典型水产养殖池塘中抗菌药物的污染特征[J].江苏农业科学,2019,47(11):210-214.
[2]Cao Y M,Lu Z J,Liu Z X.Foot-and-mouth disease vaccines:progress and problems[J].Expert Review of Vaccines,2016,15(6):783-789.
[3]Doel T R.FMD vaccines[J].Virus Research,2003,91(1):81-99.
[4]Rodriguez L.Foot-and-mouth disease virus vaccines[J].Vaccine,2011,27(S4):D90-D94.
[5]孙小涵,张碧成,张强,等.非致病性大肠杆菌鞭毛蛋白对O型口蹄疫病毒的佐剂效果[J].中国农业科学,2017,50(9):1714-1722.
[6]Sheldrake R F,Romalis L F,Saunders M M.Serum and mucosal antibody responses against Mycoplasma hyopneumoniae following intraperitoneal vaccination and challenge of pigs with M hyopneumoniae[J].Research in Veterinary Science,1993,55(3):371-376.
[7]Okamba F R,Arella M,Music N,et al.Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine[J].Vaccine,2010,28(30):4802-4809.
[8]Lee S H,Lee S W,Chae C,et al.A recombinant chimera comprising the R1 and R2 repeat regions of M.hyopneumoniae P97 and the N-terminal region of A.pleuropneumoniae Apx Ⅲ elicits immune responses[J].BMC Veterinary Research,2014,10:43.
[9]Smith K D,Andersen-Nissen E,Hayashi F,et al.Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility[J].Nature Immunology,2004,5(4):451.
[10]Hong S H,Byun Y H,Nguyen C T,et al.Intranasal administration of a flagellin-adjuvanted inactivated influenza vaccine enhances mucosal immune responses to protect mice against lethal infection[J].Vaccine,2012,30(2):466-474.
[11]Skountzou I,Martin M D,Wang B Z,et al.Salmonella flagellins are potent adjuvants for intranasally administered whole inactivated influenza vaccine[J].Vaccine,2010,28(24):4103-4112.
[12]Le Moigne V,Robreau G,Mahana W.Flagellin as a good carrier and potent adjuvant for Th1 response:study of mice immune response to the p27 (Rv2108) Mycobacterium tuberculosis antigen[J].Molecular Immunology,2008,45(9):2499-2507.
[13]Honko A N,Sriranganathan N,Lees C J,et al.Flagellin is an effective adjuvant for immunization against lethal respiratory challenge with Yersinia pestis[J].Infection and Immunity,2006,74(2):1113-1120.
[14]Albert M J,Mustafa A S,Islam A,et al.Oral immunization with cholera toxin provides protection against Campylobacter jejuni in an adult mouse intestinal colonization model[J].MBio,2013,4(3):e00213-e00246.
[15]Delaney K N,Phipps J P,Johnson J B,et al.A recombinant flagellin-poxvirus fusion protein vaccine elicits complement-dependent protection against respiratory challenge with vaccinia virus in mice[J].Viral Immunology,2010,23(2):201-210.
[16]Petrovsky N,Aguilar J C.Vaccine adjuvants:current state and future trends[J].Immunology and Cell Biology,2004,82(5):488-496.
[17]Hou J E,Liu Y,Hsi J,et al.Cholera toxin B subunit acts as a potent systemic adjuvant for HIV-1 DNA vaccination intramuscularly in mice[J].Human Vaccines & Immunotherapeutics,2014,10(5):1274-1283.
[18]Miyata T,Harakuni T,Taira T,et al.Merozoite surface protein-1 of Plasmodium yoelii fused via an oligosaccharide moiety of cholera toxin B subunit glycoprotein expressed in yeast induced protective immunity against lethal malaria infection in mice[J].Vaccine,2012,30(5):948-958.
[19]陈里新,靳玉珠,扈荣良.口蹄疫疫苗的研究进展[J].中国生物制品学杂志,2009,22(3):301-304.
[20]Gibbens J C,Sharpe C E,Wilesmith J W,et al.Descriptive epidemiology of the 2001 foot-and-mouth disease epidemic in Great Britain:the first five months[J].Veterinary Record,2001,149(24):729-743.
[21]Barate A K,Cho Y,Truong Q L,et al.Immunogenicity of IMS 1113 plus soluble subunit and chimeric proteins containing Mycoplasma hyopneumoniae P97 C-terminal repeat regions[J].FEMS Microbiology Letters,2014,352(2):213-220.
[22]陈超,李媛,郭丹,等.猪肺炎支原体p97 C末端基因重组腺病毒的构建及其免疫效果[J].微生物学报,2009,49(4):465-470.
[23]韦艳娜,熊祺琰,董璐,等.猪肺炎支原体P97R1抗原免疫刺激复合物的制备及对活疫苗免疫刺激能力的增强作用研究[J].中国预防兽医学报,2013,35(12):1016-1019.
[24]Batah J,Denève-Larrazet C,Jolivot P A,et al.Clostridium difficile flagella predominantly activate TLR5-linked NF-kappa B pathway in epithelial cells[J].Anaerobe,2016,38:116-124.
[25]Tahoun A,Jensen K,Corripio-Miyar Y,et al.Functional analysis of bovine TLR5 and association with IgA responses of cattle following systemic immunisation with H7 flagella[J].Veterinary Research,2015,46:9.
[26]Stratmann T.Cholera toxin subunit B as adjuvant-an accelerator in protective immunity and a break in autoimmunity[J].Vaccines,2015,3(3):579-596.
[27]孙小涵.非致病性大肠杆菌鞭毛蛋白增强FMDV免疫原性的研究[D].长春:吉林农业大学,2017.徐磊,孙博怿,盛鹏程,等.湖州地区典型水产养殖池塘中抗菌药物的污染特征[J].江苏农业科学,2019,47(11):210-214.