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  本文關鍵詞： 淋球菌 奈瑟菌表面蛋白A 大腸桿菌不耐熱腸毒素B亞單位 核酸疫苗 免疫活性　出處：《南華大學》2007年碩士論文　論文類型：學位論文

【摘要】： 目的:克隆淋球菌NspA基因和大腸桿菌LTB基因,用人工接頭將NspA與LTB基因融合,構建真核表達載體pcDNA3.1(-)/LTB-NspA,并用殼聚糖包裹后用作核酸疫苗;核酸疫苗通過生殖道免疫BALB/c小鼠,檢測其所誘發(fā)的體液免疫(特別是粘膜免疫)和細胞免疫應答水平,為研制新型、高效的淋球菌核酸疫苗提供實驗依據。 方法:分別以淋球菌WHO-A株的NspA核酸序列和大腸桿菌H44815株LTB核酸序列為模板,PCR擴增NspA全基因和LTB基因;同時以編碼6個氨基酸的18個寡核苷酸作為接頭,通過重組PCR法將NspA與LTB基因融合,PCR產物經酶切純化后克隆至pcDNA3.1(-)真核表達載體中;陽性克隆經雙酶切及測序鑒定,大量制備質粒純化后作為粘膜核酸疫苗。同時構建原核表達重組體pET-30a/NspA、pET-30a/LTB及pET-30a/LTB-NspA,在E.coli BL21中表達重組蛋白。以殼聚糖包裹的pcDNA3.1(-)/NspA和pcDNA3.1(-)/LTB-NspA生殖道免疫4w齡BALB/c小鼠,免疫熒光組化法檢測淋球菌NspA基因和大腸桿菌LTB基因在小鼠生殖道粘膜內的表達,間接ELISA法檢測免疫小鼠生殖道粘膜分泌液中抗NspA的sIgA抗體水平和血清中抗NspA的IgG抗體水平,ELISA雙抗體夾心法檢測脾淋巴細胞培養(yǎng)上清中IFN-γ水平,MTT比色法檢測脾淋巴細胞增殖反應。 結果:成功構建了pcDNA3.1(-)/NspA和pcDNA3.1(-)/LTB-NspA真核表達載體,以殼聚糖包裹后作為疫苗通過生殖道免疫小鼠后,生殖道粘膜組織免疫熒光法檢測到了NspA、LTB蛋白的表達。末次免疫后小鼠粘膜分泌液中檢測到了抗NspA的sIgA抗體,其水平隨免疫時間延長而增高,并且含粘膜佐劑的核酸疫苗pcDNA3.1(-)/LTB-NspA免疫組小鼠sIgA水平明顯高于免疫不含粘膜佐劑的核酸疫苗pcDNA3.1(-)/NspA組小鼠(P0.05)。兩核酸疫苗免疫組小鼠血清中均產生了一定水平的IgG抗體,兩組間IgG水平無明顯差異(P0.05)。核酸疫苗免疫組小鼠脾淋巴細胞經PHA刺激后,培養(yǎng)上清中IFN-γ含量明顯升高[pcDNA3.1(-)/NspA組達135.86±13.97pg/mL,pcDNA3.1(-)/LTB-NspA組達140.18±20.54pg/mL],與空質粒組(45.24±5.31pg/mL)和PBS組(5.75±1.12pg/mL)之間有顯著性差異(P0.01),兩核酸疫苗免疫組間無明顯差異(P0.05)。脾淋巴細胞增殖反應測定,核酸疫苗pcDNA3.1(-)/LTB-NspA組和pcDNA3.1(-)/NspA組小鼠脾淋巴細胞經PHA刺激后,刺激指數分別為(1.573±0.012)和(1.518±0.010),明顯高于空質粒組(1.134±0.007)和PBS組(1.044±0.005) (P0.01),兩核酸疫苗免疫組間無明顯差異(P0.05)。結論: (1)成功克隆了淋球菌NspA基因和大腸桿菌LTB基因,并構建了pcDNA3.1(-)/NspA和含粘膜佐劑的pcDNA3.1(-)/LTB-NspA核酸疫苗。 (2)成功構建了原核表達重組體pET-30a/NspA、pET-30a/LTB及pET-30a/ LTB-NspA,重組蛋白NspA和LTB-NspA在E.coli BL21中獲得了表達。 (3)殼聚糖包裹的核酸疫苗pcDNA3.1(-)/NspA和pcDNA3.1(-)/LTB-NspA經生殖道免疫BALB/c小鼠后,能被粘膜攝取并在粘膜組織內獲得表達。 (4)殼聚糖包裹的核酸疫苗經生殖道免疫小鼠后,誘發(fā)了一定水平的體液免疫(特別是粘膜免疫)和細胞免疫應答;含粘膜佐劑的核酸疫苗pcDNA3.1(-)/LTB-NspA較不含粘膜佐劑的核酸疫苗pcDNA3.1(-)/NspA所誘發(fā)的粘膜免疫水平高;而IgG水平和細胞免疫應答水平二者差異不明顯。
[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 .

【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

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