呼吸道合胞病毒G蛋白基因重组质粒诱导小鼠免疫保护性及机制研究

目的 构建编码呼吸道合胞病毒(RSV)G蛋白基因的重组质粒,观察其对RSV感染小鼠的保护性及特异性免疫效应,为研制安全有效的RSV新疫苗提供线索和实验依据.方法 利用基因重组方法构建含RSV G蛋白编码基因的重组质粒,测序和酶切鉴定,Western blot检测目的 基因经体外表达后的免疫原性.重组质粒免疫小鼠后以RSV感染,病毒滴定法检测肺组织标本中RSV滴度,HE染色法检查肺组织病理改变,ELISA检测血清抗体水平,双抗体夹心ELISA测定肺泡灌洗液(BALF)内Th1/Th2细胞因子表达量,流式细胞术检测BALF中T淋巴细胞亚群数量及活化状态.结果 成功构建了编码RSV G蛋白基因的重组质粒pcDNA3.1~G.Western blot证实目的 蛋白在体外具有免疫原性.被免疫小鼠感染RSV后肺组织病毒滴度降低,肺组织中未见明显的炎性细胞浸润.小鼠血清中产生较高滴度的抗RSV-G IgG.小鼠BALF细胞中CD4~+CD25~+T淋巴细胞比例显著增加,并且IFN-γ(Th1)、IL-4(Th2)两类细胞因子表达显示平衡.结论 编码RSV G蛋白基因的重组质粒pcDNA3.1~G能诱导产生CD4~+ CD25~+T细胞亚群,对小鼠具有明显的保护作用.

Immune protection and mechanism of plasmid DNA encoding Gglycoprotein of respiratory syncytial virus(RSV)

Objective To construct a plasmid DNA encoding G glycoprotein of respiratory syncytial virus(RSV) and investigate the protective immune response against RSV infection. Methods Recombinant plasmid DNA of pcDNA3.1~G was constructed by standard RT-PCR based cloning procedure. The immunogenicity of recombinant G protein transiently expressed in HEK293 cells was detected by Western blot. BABL/c mice were intramuscularly immunized with pcDNA3.1~G. Samples of lung, sera, bronchoalveolar lavage fluid(BALF) were collected before and after RSV challenge; virus titer in lung was detected by viral titration; sections of paraffin embedding lung tissues were stained by haematoxylin and eosin(HE) for histological analyses; sera anti-RSV IgG levels were examined by ELISA; Th1/Th2 cytokine were detected by ELISA kit, the T lymphocyte subsets of BALF was determined by immunefluorescence staining followed by flow cytometry. Results Plasmid DNA of pcDNA3.1~G was successfully constructed. The expressed target protein possesses immunogenicity. After challenge, pcDNA3.1~G immunized mice presented relieved pathological changes in lung as well as reduced lung viral titers. The RSV specific IgG was detected in sera of immunized mice. There was significantly increased number of CD25~+CD4~+ T cells in mice BALF. Conclusion We constructed a pcDNA3.1~G plasmid DNA vaccination which can induce evident protective cellular immunity against RSV infection in mice with the increased number of CD25~+CD4~+ T cell subpopulation.