摘要
目的考察热休克蛋白HSP70对呼吸道合胞病毒(RSV)重组蛋白抗原G1F/M2免疫应答的调节作用。方法构建并表达了Javelin-G1F/M2重组蛋白,在G1F/M2的N端引入Javelin序列,通过该序列可以实现重组蛋白与HSP70蛋白高亲和力结合。以HSP70:Javelin-G1F/M2复合物等免疫Balb/c小鼠,用乳酸脱氢酶释放法测定CTL活性,ELISA法测定IgG及其亚型IgG1和IgG2a抗体滴度,空斑抑制实验测定血清中和抗体。结果经表达和纯化获得了纯度达90%以上的融合蛋白;皮下免疫HSP70:Javelin-G1F/M2复合物可诱导高滴度的IgG抗体和强的CTL应答,其抗体滴度和CTL应答强度均优于Javelin-G1F/M2加铝佐剂腹腔免疫和单独Javelin-G1F/M2皮下免疫。HSP70:Javelin-G1F/M2复合物在诱导中和抗体方面与Javelin-G1F/M2加铝佐剂腹腔免疫基本相当。HSP70:Javelin-G1F/M2复合物可以诱导更强的IgG2a应答,IgG的亚型IgG1/IgG2a的比值明显低于Javelin-G1F/M2加铝佐剂腹腔免疫组和单独Javelin-G1F/M2皮下免疫组。结论 HSP70可能通过增强CTL应答进一步纠正Th2型优势应答,使得Th1/Th2应答更加平衡。
This study aims to construct a new recombinant immunogen Javelin-G1F/M2 which can bind to HSP70 in high-affinity and investigate the regulatory effects of HSPs on the immunological response of recombinant protein immunogen G1F/M2.By primers design and PCR amplification,peptide Javelin was introduced to the N terminal of G1F/M2.The recombinant protein Javelin-G1F/M2 was expressed successfully in E.coli and the purity was over 90% after purification by affinity chromatography.Balb/c mice were immunized intraperitoneally with Javelin-G1F/M2 in 20% alhydrogel or immunized subcutaneously with HSP70：Javelin-G1F/M2 complex or Javelin-G1F/M2 without adjuvant.Serum samples of mice were collected 10 days after the last immunization.CTL responses were measured using a Lactate Dehydrogenase（LDH） Cytotoxicity Assay Kit;IgG,IgG1,and IgG2a antibodies were detected by ELISA;and neutralizing antibodies were assayed by plaque reduction assay.High titer of IgG antibody and strong CTL responses were induced in Balb/c mice subcutaneously vaccinated with HSP70：Javelin-G1F/M2 complex without additional adjuvant.The titer of IgG antibody and the activity of CTL induced by HSP70：Javelin-G1F/M2 complex were markedly higher than those in mice subcutaneously vaccinated with Javelin-G1F/M2 or intraperitoneal vaccinated with Javelin-G1F/M2 formulated in Al（OH）3.The titer of neutralization antibody induced by HSP70：Javelin-G1F/M2 was equivalent with that induced by Javelin-G1F/M2 formulated in Al（OH）3.Compared with Javelin-G1F/M2 formulated in Al（OH）3,HSP70：JavelinG1F/M2 complex can induce more Th1/Th2 response in balance.The above results indicated the CTL response of antigen G1F/M2 can be enhanced by HSP70 in forms of HSP70：G1F/M2 complex,thus the ratio of IgG1/IgG2a is downregulated and more balanced Th1/Th2 responses are induced.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2012年第1期5-9,39,共6页
Immunological Journal
基金
国家自然科学基金项目(30671923)
作者简介
通信作者：梅兴国，Tel：010—66932644：E—mail：newdds@126．com
