CAJ | 학술논문

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.
목적 구건편마호흡도합포병독(RSV)G단백기인적중조질립,관찰기대RSV감염소서적보호성급특이성면역효응,위연제안전유효적RSV신역묘제공선색화실험의거.방법 이용기인중조방법구건함RSV G단백편마기인적중조질립,측서화매절감정,Western blot검측목적 기인경체외표체후적면역원성.중조질립면역소서후이RSV감염,병독적정법검측폐조직표본중RSV적도,HE염색법검사폐조직병리개변,ELISA검측혈청항체수평,쌍항체협심ELISA측정폐포관세액(BALF)내Th1/Th2세포인자표체량,류식세포술검측BALF중T림파세포아군수량급활화상태.결과 성공구건료편마RSV G단백기인적중조질립pcDNA3.1~G.Western blot증실목적 단백재체외구유면역원성.피면역소서감염RSV후폐조직병독적도강저,폐조직중미견명현적염성세포침윤.소서혈청중산생교고적도적항RSV-G IgG.소서BALF세포중CD4~+CD25~+T림파세포비례현저증가,병차IFN-γ(Th1)、IL-4(Th2)량류세포인자표체현시평형.결론 편마RSV G단백기인적중조질립pcDNA3.1~G능유도산생CD4~+ CD25~+T세포아군,대소서구유명현적보호작용.
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.