【作者】 马艳琴；

【导师】 聂向庭； 张映；

【作者基本信息】 山西农业大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 CpG基序是以未甲基化CpG二核苷酸为核心的寡聚脱氧核苷酸，其结构特征为：5’嘌呤-嘌呤-CpG-嘧啶-嘧啶3’。实验证明，人工合成的含CpG基序的寡脱氧核苷酸(CpG ODN)可以模拟细菌、病毒的自然危险信号，诱发机体产生强烈的Th1型免疫应答，有望成为一类新型的免疫佐剂。进一步的研究表明，哺乳动物对CpG ODN的识别具有种属特异性。本实验致力于研究体外对猪免疫细胞具有刺激活性的CpG ODN的结构特点，为进一步研究适用于猪的疫苗佐剂提供实验参考。 依据相关资料，设计并合成了38条CpG ODN。应用淋巴细胞增殖实验(LPR)和IFN-γ分泌实验来评价体外CpG ODN对猪外周血单个核细胞(PBMC)的免疫刺激活性。采用外周血单个核细胞培养、[~3H]-脱氧胸苷掺入检测技术，以刺激指数(SI值)为检测指标，比较了不同的CpG ODN刺激猪PBMC增殖的作用。发现具有免疫刺激作用的CpG ODN具有如下结构特点：①CpG ODN的作用依赖于序列中CpG二核苷酸的存在；②长度在20～25bp、含有三个或四个基序为最适序列；③ODN序列的磷酸戊糖骨架修饰对其作用也有影响。采用双抗体夹心ELISA检测了PBMC培养上清液中的IFN-γ的含量，发现能有效刺激猪PBMC分泌IFN-γ的序列是：以“嘌呤-嘧啶-CG-嘌呤-嘧啶”基序为中心，有一定的侧翼序列，并形成一种互补的回文结构；在3’端和5’端还加有poly G尾；为磷酸二酯键(PO)／硫代磷酸二酯键(PTO)混合型骨架，中心的碱基由磷酸二酯键连接，两端的poly G尾由硫代磷酸二酯键连接。在研究中还发现，能刺激猪PBMC增殖的CpG ODN和刺激其分泌IFN-γ的CpG ODN为两类结构不同的ODN。该实验结果为进一步研究适用于不同病原体的猪用疫苗佐剂奠定了基础。更多还原

【Abstract】 CpG motif is a 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5’purines and two 3’pyrimidines. Synthetic Oligodeoxynucleotides (ODN) containing CpG motif were demonstrated to mimic a danger signal of bacterial or viral DNA and induce a potent Th1 biased immune response. Therefore, CpG ODN may have potential as a new adjuvant. Further studies documented that different motifs are optimally stimulatory in different species. The current work examines the structure character of the ODN which can stimulate porcine immune cells in vitro and provides an experimental foundation for intensive study on vaccine adjuvant for swine.Using lymphocyte proliferative response (LPR) and IFN- Y secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on porcine peripheral blood mononuclear cells (PBMC) . The PBMC were cultured with ODN and the stimulation index was determined by 3H-TdR incorporation assay. Results show that the immunostimulatory CpG ODN has the following structure: (1)The CpG dinucleotide is needed; (2)The oligonucleotide is optimal which contains 3 or 4 motifs and whose length is between 20-25bp; (3)The modification of the backbone of the CpG ODN has a prominent influence on its immunostimulatory activity. The amount of IFN-γ in the culture supernatant was measured by swIFN-γ kit which is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA) . This work demonstrates that the ODN which can stimulate porcine PBMC to secrete IFN- γ has the following structure : It has mixed phosphodiester/phosphorothioate backbone and contains a single hexameric purine/pyrimidine/CG/purine/pyrimidine motif flanked by self-complementary bases that form a stem-loop structure capped at the 3’ and 5’ end by a polyG tail. These findings indicate that porcine PBMC recognize and respond to two structurally distinct classes of CpG ODN. One class preferentially induces cell proliferation, whereas the other stimulates IFN- γ release. The distinct immunostimulatory properties of the two classes of CpG ODN improve the design of CpG ODN as vaccine adjuvants.更多还原