分子佐剂鞭毛素对弓形虫SAG1蛋白抗体应答的影响
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摘要
将沙门氏菌鞭毛素和弓形虫免疫优势表面抗原1(SAG1)融合表达,研究其在小鼠上激发的体液免疫应答.首先,采用PCR扩增SAG1片段.其次,将SAG1连接到原核表达载体pET-28a中构建表达质粒pET-28a-SAG1;连接到实验室已有的pET-28a鞭毛素质粒中构建表达质粒pET-28a-F-SAG1;质粒转化至大肠杆菌BL21(DE3)菌株中并进行表达.将纯化后的蛋白免疫小鼠,每隔2周免疫一次,共免疫3次,第3次免疫后15 d取小鼠血清.最后,采用蛋白印迹检测蛋白的大小;用酶联免疫吸附方法检测血清中多克隆抗体的效价.结果表明,诱导出的SAG1蛋白大小为30 ku,F-SAG1大小为70 ku.酶联免疫吸附方法检测的D450 nm均为阴性对照的两倍,表明该多克隆抗体具有较高效价.可见,鞭毛素可以作为分子佐剂促进弓形虫亚单位疫苗的体液免疫应答.
Flagellin,a ligand for TLR5,can be an effective adjuvant for vaccine. In this study,we coexpressed the flagellin and Toxoplasma gondii immune dominant antigen SAG1(surface antigen 1),the immunogenicity of SAG1 and adjuvant property of flagellin for SAG1 protein in mice were investigated. SAG1 was amplified by PCR,and inserted into prokaryotic expression vector to construct expression plasmid pET-28a-SAG1. Then SAG1 was inserted into pET-28a-F to construct expression plasmid pET-28a-FSAG1. Subsequently,these 2 plasmids were transformed into BL21(DE3) E.coli and expressed. Purified proteins were intramuscularly injected with the same amount of adjuvant in mice,and immuned at an interval of 2 weeks. After 3 immunization,the serum was obtained and the titer of the polyclonal antibody was evaluated by ELISA. The results showed that SAG1 protein was 30 ku and F-SAG1 was 70 ku. ELISA D450 nmvalues were twice more than the negative control,indicating a high titer of the polyclonal antibody.In conclusion,flagellin can be an effective adjuvant to promote the humoral immunity of subunit vaccine.
Flagellin,a ligand for TLR5,can be an effective adjuvant for vaccine. In this study,we coexpressed the flagellin and Toxoplasma gondii immune dominant antigen SAG1(surface antigen 1),the immunogenicity of SAG1 and adjuvant property of flagellin for SAG1 protein in mice were investigated. SAG1 was amplified by PCR,and inserted into prokaryotic expression vector to construct expression plasmid pET-28a-SAG1. Then SAG1 was inserted into pET-28a-F to construct expression plasmid pET-28a-FSAG1. Subsequently,these 2 plasmids were transformed into BL21(DE3) E.coli and expressed. Purified proteins were intramuscularly injected with the same amount of adjuvant in mice,and immuned at an interval of 2 weeks. After 3 immunization,the serum was obtained and the titer of the polyclonal antibody was evaluated by ELISA. The results showed that SAG1 protein was 30 ku and F-SAG1 was 70 ku. ELISA D450 nmvalues were twice more than the negative control,indicating a high titer of the polyclonal antibody.In conclusion,flagellin can be an effective adjuvant to promote the humoral immunity of subunit vaccine.
引文
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[12]REICHHART J M.TLR5 takes aim at bacterial propeller[J].Nature Immunology,2003,4(12):1 159-1 160.
[13]CARON G,DULUC D,FRMAUX I,et al.Direct stimulation of human T cells via TLR5 and TLR7/8:Flagellin and R-848up-regulate proliferation and IFN-γproduction by memory CD4+T cells[J].Journal of Immunology,2005,175(3):1 551-1 557.
[14]潘杭君,孙红祥.免疫佐剂的研究进展[J].中国兽药杂志,2004,38(1):32-37.
[15]HUIJBERS E J M,FEMEL J,ANDERSSON K,et al.The non-toxic and biodegradable adjuvant Montanide ISA 720/Cp G can replace Freund's in a cancer vaccine targeting ED-B—a prerequisite for clinical development[J].Vaccine,2012,30(2):225-230.
[16]游猛,潘志明,耿士忠,等.鼠伤寒沙门菌鞭毛蛋白与新城疫病毒F蛋白的融合表达及免疫原性分析[J].动物医学进展,2010,31(2):20-25.
[17]TALLANT T,DEB A,KAR N,et al.Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells[J].BMC Microbiology,2004,4(1):33.
[2]ZOU F C,SUN X T,XIE Y J,et al.Seroprevalence of Toxoplasma gondii in pigs in southwestern China[J].Parasitology International,2009,58(3):306-307.
[3]张念章.弓形虫CDPKs核酸疫苗及重组亚单位疫苗的免疫保护性研究[D].北京:中国农业科学院,2014.
[4]DODDS E M.Toxoplasmosis[J].Current Opinion in Ophthalmology,2006,17(6):557-561.
[5]PENG Z,CHEN Z,LI H L,et al.Toxoplasma gondii,infection in humans in China[J].Parasites&Vectors,2011,4(1):1-9.
[6]SMITH A T,TUCKERSAMARAS S D,FAIRLAMB A H,et al.MYST family histone acetyltransferases in the protozoan parasite Toxoplasma gondii[J].Eukaryotic Cell,2005,4(12):2 057-2 065.
[7]蒲元华,王艳华,王萌,等.抗弓形虫药物及机理研究进展[J].中国人兽共患病学报,2012,28(4):389-392.
[8]ZHANG N Z,CHEN J,WANG M,et al.Vaccines against Toxoplasma gondii:new developments and perspectives[J].Expert Review of Vaccines,2013,12(11):1 287-1 299.
[9]朱思梅,周亚萍,朱荫昌,等.弓形虫表面抗原SAG1的可溶性表达及其免疫学活性分析[J].中国病原生物学杂志,2011,6(11):816-818,812.
[10]JONGERT E,ROBERTS C W,GARGANO N,et al.Vaccines against Toxoplasma gondii:challenges and opportunities[J].Mem Inst Oswaldo Cruz,2009,104(2):252-266.
[11]李瑾,肖婷,黄炳成,等.弓形虫pc DNA3-P30-ROP2-HSP70复合核酸疫苗免疫保护性的研究[J].中国人兽共患病学报,2012,28(1):56-62.
[12]REICHHART J M.TLR5 takes aim at bacterial propeller[J].Nature Immunology,2003,4(12):1 159-1 160.
[13]CARON G,DULUC D,FRMAUX I,et al.Direct stimulation of human T cells via TLR5 and TLR7/8:Flagellin and R-848up-regulate proliferation and IFN-γproduction by memory CD4+T cells[J].Journal of Immunology,2005,175(3):1 551-1 557.
[14]潘杭君,孙红祥.免疫佐剂的研究进展[J].中国兽药杂志,2004,38(1):32-37.
[15]HUIJBERS E J M,FEMEL J,ANDERSSON K,et al.The non-toxic and biodegradable adjuvant Montanide ISA 720/Cp G can replace Freund's in a cancer vaccine targeting ED-B—a prerequisite for clinical development[J].Vaccine,2012,30(2):225-230.
[16]游猛,潘志明,耿士忠,等.鼠伤寒沙门菌鞭毛蛋白与新城疫病毒F蛋白的融合表达及免疫原性分析[J].动物医学进展,2010,31(2):20-25.
[17]TALLANT T,DEB A,KAR N,et al.Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells[J].BMC Microbiology,2004,4(1):33.