大肠埃希菌肠毒素B亚基对弓形虫速殖子SAG1-ROP2复合基因免疫效果的影响

目的 研究大肠埃希菌不耐热肠毒素B亚基（LTB）对弓形虫速殖子SAG1-ROP2复合基因免疫效果的影响。 方法 构建重组真核表达质粒pcDNA3.1-SAG1-ROP2和pEASY-E1-LTB。BALB/c小鼠88只，随机均分为4组，分别用PBS（A组）、 pcDNA3.1空质粒（B组）、 pcDNA3.1-SAG1-ROP2质粒（C组），以及pcDNA3.1-SAG1-ROP2质粒和pEASY?-E1-LTB质粒（D组）进行滴鼻免疫，每种质粒20 μg/（只·次）。每组小鼠随机抽取15只，每周免疫1次，共4次，末次免疫后2周，测定其血清IgG和IgA抗体水平，气管和小肠黏膜冲洗液分泌型IgA（sIgA）水平，以及脾细胞培养上清中γ干扰素（IFN-γ）和白细胞介素4（IL-4）水平；每组中余7只小鼠，每周免疫1次，共3次，末次免疫后4周，弓形虫速殖子腹腔接种感染（1×103/鼠），观察比较各组生存时间。 结果 成功构建pEASY-E1-LTB重组表达质粒。D组小鼠血清IgG(0.626±0.100)和IgA抗体水平(1.086±0.138)，气管和小肠黏膜冲洗液sIgA水平(0.886±0.164)，以及细胞因子IFN-γ[(2 017±266)pg/ml]和IL-4水平[(203±31)pg/ml]均显著高于其他各组（均P<0.05）。感染弓形虫速殖子后，A、B、C和D组小鼠的生存时间中位数分别为3、4、6和10 d，D组的生存时间长于其他各组（均P<0.05）。 结论 LTB能明显增强弓形虫速殖子SAG1?鄄ROP2复合基因的免疫效果。     

内置式佐剂LTB对空肠弯曲菌重组蛋白疫苗免疫增强作用的研究

目的 构建空肠弯曲菌(CJ)外膜蛋白(PEB1)及大肠埃希菌LTB融合疫苗并研究其免疫原性.方法 应用重组PCR技术将PEB1和LTB序列连接,构建PEB1-LTB融合基因,将其克隆入原核表达载体pET-28a,转化入大肠埃希菌BL21,SDS- PAGE电泳表达,Western印迹检测表达蛋白的抗原性.肌注方式免疫小鼠,末次免疫2周后,测定小鼠血清中IgG、IgA及黏膜冲洗液中sIgA抗体水平.结果 PCR扩增分别获得约787、372 bp产物,二者融合后获得一约1180 bp目的基因；该重组蛋白可具有良好的免疫原性,肌注小鼠后其血清IgG、IgA及黏膜冲洗液中sIgA含量均显著增高(P ＜0.01).结论 构建PEB1-LTB融合蛋白可有效增强小鼠免疫应答水平.     

LT-IIc, a new member of the type II heat-labile enterotoxin family, exhibits potent immunomodulatory properties that are different from those induced by LT-IIa or LT-IIb

A plethora of human pathogens invade and/or colonize mucosal surfaces. Elaboration of strong, protective immune responses against those pathogens by mucosal vaccination, however, is hampered by endogenous regulatory systems in the mucosae that dampen responses to foreign antigens (Ags). To overcome those natural barriers, mucosal adjuvants must be employed. Using a mouse mucosal immunization model and AgI/II, a weak immunogen from Streptococcus mutans, LT-IIc, a new member of the type II subgroup of the heat-labile enterotoxin family, was shown to have potent mucosal adjuvant properties. In comparison to mice intranasally immunized only with AgI/II, co-administration of AgI/II with LT-IIc enhanced production of Ag-specific IgA antibodies in the saliva and vaginal fluids and Ag-specific IgA and IgG in the serum. Secretion of IL-2, IL-6, IL-17, IFN-γ, and TNF-α was enhanced in cultures of AgI/II-stimulated splenic cells isolated from mice that had received LT-IIc as a mucosal adjuvant. In contrast, secretion of IL-10 was suppressed in those cells. This pattern of cytokine secretion suggested that LT-IIc stimulates both Th1 and Th2 immune responses. In contrast to LT-IIa and LT-IIb, the original members of the type II subgroup that also are mucosal adjuvants, LT-IIc dramatically enhanced secretion of IL-1α and IL-1β in peritoneal macrophages that had been co-cultured with LPS. Furthermore, the B pentameric subunit of LT-IIc augmented uptake of Ag by bone marrow-derived dendritic cells to levels that exceeded those attained by use of LPS or by the B pentamers of LT-IIa or LT-IIb. These data confirmed that LT-IIc is a strong mucosal adjuvant with immunomodulatory properties that are distinguishable from those of LT-IIa and LT-IIb and which has immunomodulatory properties that may be exploitable in vaccine development.     

空肠弯曲菌PEB1-LTB DNA疫苗构建及其对小鼠免疫保护性的研究

目的:研究粘膜佐剂大肠埃希菌不耐热肠毒素B亚单位(LTB)辅助的空肠弯曲菌(C.jejuni)外膜蛋白PEB1基因重组DNA疫苗诱导小鼠免疫应答水平。方法:构建pcDNA3.1(-)-PEB1-LTB真核重组表达载体,转染Hela细胞,Westernblot鉴定蛋白表达。滴鼻免疫BALB/c小鼠,末次免疫后2周,测定小鼠血清中IgG、IgA及气管、小肠粘膜冲洗液sIgA抗体;脾细胞培养上清中IFN-γ、IL-4水平。末次免疫后4周,采用空肠弯曲菌重复攻击的方式进行灌胃攻击,攻击后,根据动物疾病指数评价疫苗临床保护率。结果:构建的重组表达质粒能在Hela细胞内表达,重组蛋白。疫苗免疫小鼠后,不仅诱导了高水平血清IgG、IgA抗体,而且诱导了高水平粘膜sIgA抗体。其诱导的特异性免疫应答能有效保护免疫后小鼠免遭空肠弯曲菌的感染攻击。结论:构建的DNA疫苗经粘膜免疫能诱导小鼠产生较高水平的特异性免疫应答,能有效预防空肠弯曲菌感染。     

含扩增内对照的霍乱毒素基因ctx和不耐热肠毒素基因elt三重real-time PCR检测体系的建立

目的 建立一个含扩增内对照（IAC）的三重TaqMan real-time PCR体系，以检测霍乱毒素基因ctxA 和肠产毒性大肠埃希菌的不耐热肠毒素基因elt。方法 针对ctxA、elt 和IAC设计引物和探针，进行灵敏性和特异性分析，评价三重反应之间的互相影响。结果 该检测体系灵敏度为ctxA 每个反应94 拷贝，elt 每个反应79 拷贝，扩增效率分别为94.7%与98.1%。ctxA 与elt 拷贝数比例为1 ∶ 1～1 ∶ 10 时，二者均能良好扩增;elt 或ctxA 的量是IAC的100 倍以上时，IAC扩增受到抑制。结论 该检测体系具有良好的灵敏性和特异性，可以用于腹泻粪便中感染病原菌的检测，其中内对照检测可以提示粪便样本中是否存在PCR抑制因子。