淋球菌NspA-LTB核酸疫苗的构建及其免疫活性的初步研究

发布时间：2018-01-19 04:32

  本文关键词： 淋球菌 奈瑟菌表面蛋白A 大肠杆菌不耐热肠毒素B亚单位 核酸疫苗 免疫活性　出处：《南华大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:克隆淋球菌NspA基因和大肠杆菌LTB基因,用人工接头将NspA与LTB基因融合,构建真核表达载体pcDNA3.1(-)/LTB-NspA,并用壳聚糖包裹后用作核酸疫苗;核酸疫苗通过生殖道免疫BALB/c小鼠,检测其所诱发的体液免疫(特别是粘膜免疫)和细胞免疫应答水平,为研制新型、高效的淋球菌核酸疫苗提供实验依据。 方法:分别以淋球菌WHO-A株的NspA核酸序列和大肠杆菌H44815株LTB核酸序列为模板,PCR扩增NspA全基因和LTB基因;同时以编码6个氨基酸的18个寡核苷酸作为接头,通过重组PCR法将NspA与LTB基因融合,PCR产物经酶切纯化后克隆至pcDNA3.1(-)真核表达载体中;阳性克隆经双酶切及测序鉴定,大量制备质粒纯化后作为粘膜核酸疫苗。同时构建原核表达重组体pET-30a/NspA、pET-30a/LTB及pET-30a/LTB-NspA,在E.coli BL21中表达重组蛋白。以壳聚糖包裹的pcDNA3.1(-)/NspA和pcDNA3.1(-)/LTB-NspA生殖道免疫4w龄BALB/c小鼠,免疫荧光组化法检测淋球菌NspA基因和大肠杆菌LTB基因在小鼠生殖道粘膜内的表达,间接ELISA法检测免疫小鼠生殖道粘膜分泌液中抗NspA的sIgA抗体水平和血清中抗NspA的IgG抗体水平,ELISA双抗体夹心法检测脾淋巴细胞培养上清中IFN-γ水平,MTT比色法检测脾淋巴细胞增殖反应。 结果:成功构建了pcDNA3.1(-)/NspA和pcDNA3.1(-)/LTB-NspA真核表达载体,以壳聚糖包裹后作为疫苗通过生殖道免疫小鼠后,生殖道粘膜组织免疫荧光法检测到了NspA、LTB蛋白的表达。末次免疫后小鼠粘膜分泌液中检测到了抗NspA的sIgA抗体,其水平随免疫时间延长而增高,并且含粘膜佐剂的核酸疫苗pcDNA3.1(-)/LTB-NspA免疫组小鼠sIgA水平明显高于免疫不含粘膜佐剂的核酸疫苗pcDNA3.1(-)/NspA组小鼠(P0.05)。两核酸疫苗免疫组小鼠血清中均产生了一定水平的IgG抗体,两组间IgG水平无明显差异(P0.05)。核酸疫苗免疫组小鼠脾淋巴细胞经PHA刺激后,培养上清中IFN-γ含量明显升高[pcDNA3.1(-)/NspA组达135.86±13.97pg/mL,pcDNA3.1(-)/LTB-NspA组达140.18±20.54pg/mL],与空质粒组(45.24±5.31pg/mL)和PBS组(5.75±1.12pg/mL)之间有显著性差异(P0.01),两核酸疫苗免疫组间无明显差异(P0.05)。脾淋巴细胞增殖反应测定,核酸疫苗pcDNA3.1(-)/LTB-NspA组和pcDNA3.1(-)/NspA组小鼠脾淋巴细胞经PHA刺激后,刺激指数分别为(1.573±0.012)和(1.518±0.010),明显高于空质粒组(1.134±0.007)和PBS组(1.044±0.005) (P0.01),两核酸疫苗免疫组间无明显差异(P0.05)。结论: (1)成功克隆了淋球菌NspA基因和大肠杆菌LTB基因,并构建了pcDNA3.1(-)/NspA和含粘膜佐剂的pcDNA3.1(-)/LTB-NspA核酸疫苗。 (2)成功构建了原核表达重组体pET-30a/NspA、pET-30a/LTB及pET-30a/ LTB-NspA,重组蛋白NspA和LTB-NspA在E.coli BL21中获得了表达。 (3)壳聚糖包裹的核酸疫苗pcDNA3.1(-)/NspA和pcDNA3.1(-)/LTB-NspA经生殖道免疫BALB/c小鼠后,能被粘膜摄取并在粘膜组织内获得表达。 (4)壳聚糖包裹的核酸疫苗经生殖道免疫小鼠后,诱发了一定水平的体液免疫(特别是粘膜免疫)和细胞免疫应答;含粘膜佐剂的核酸疫苗pcDNA3.1(-)/LTB-NspA较不含粘膜佐剂的核酸疫苗pcDNA3.1(-)/NspA所诱发的粘膜免疫水平高;而IgG水平和细胞免疫应答水平二者差异不明显。
[Abstract]:Objective : To clone gonococcus NspA gene and E . coli LTB gene , and to construct eukaryotic expression vector pcDNA3.1 ( - ) / LTB - NspA by artificial joint . The eukaryotic expression vector pcDNA3.1 ( - ) / LTB - NspA was constructed and coated with chitosan as a nucleic acid vaccine . Methods : The NspA gene and LTB gene were amplified by PCR . The recombinant protein was cloned into pcDNA3.1 ( - ) eukaryotic expression vector by recombinant PCR . The recombinant pET - 30a / NspA , pET - 30a / LTB and pET - 30a / LTB - NspA were cloned into the eukaryotic expression vector pcDNA3.1 ( - ) / LTB - NspA , pET - 30a / LTB and pET - 30a / LTB - NspA . Results : The recombinant pcDNA3.1 ( - ) / NspA and pcDNA3.1 ( - ) / LTB - NspA were significantly higher than that of pcDNA3.1 ( - ) / NspA group ( P0.05 ) . ( 1 ) The NspA gene and LTB gene of Escherichia coli were cloned successfully , and pcDNA3.1 ( - ) / NspA and pcDNA3.1 ( - ) / LTB - NspA nucleic acid vaccine containing mucosal adjuvant were constructed . ( 2 ) The prokaryotic expression recombinant pET - 30a / NspA , pET - 30a / LTB and pET - 30a / LTB - NspA were constructed successfully . The recombinant protein NspA and LTB - NspA were expressed in E . coli BL21 . ( 3 ) The chitosan - coated nucleic acid vaccine pcDNA3.1 ( - ) / NspA and pcDNA3.1 ( - ) / LTB - NspA were immune to BALB / c mice by reproductive tract , and can be taken up by mucosa and expressed in mucosal tissues . ( 4 ) After immunized with chitosan - coated nucleic acid vaccine , a certain level of humoral immunity ( especially mucosal immunity ) and cellular immune response were induced . The mucosal immune level induced by pcDNA3.1 ( - ) / LTB - NspA of nucleic acid vaccine containing mucosal adjuvant was higher than that of pcDNA3.1 ( - ) / NspA without mucosal adjuvant .

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【参考文献】

相关期刊论文 前4条

1 邵长庚;性传播疾病的流行现状及其防治对策[J];国外医学.皮肤性病学分册;1995年03期

2 董志炜,潘善培,谢琪璇,肖銮娟,孙彩军,余巍,何柳媚;小鼠口服猪卵透明带DNA避孕疫苗pVAX1-pZP3α的抗生育研究[J];暨南大学学报(自然科学与医学版);2004年06期

3 何芳,赵连三;淋病疫苗研制的若干问题[J];中华预防医学杂志;1998年04期

4 李学荣,余新炳;核酸疫苗及其免疫机制研究[J];中国人兽共患病杂志;2000年06期

，

本文编号：1442671