ECMS-oil对兔波氏杆菌疫苗的佐剂作用及机理研究

Rabbit Bordetella bronchiseptica（Bb） is one of important respiratory infectious diseases in rabbit. The disease induced huge economic losses. The immune effects of present bordetella bronchiseptica vaccine were not ideal. In order to find a safe and effective vaccine adjuvant, ECMS-oil（extraction of Cochinchina momordica seed-oil） will be studied for its adjuvant activity on immune responses against Bordetella bronchiseptica. Total and subclass of antibody in rabbit serum against Bb after immunization will be measured by ELISA methods. Concentrations of cytokines from lymphocytes supernatant, the expression of cytokines or transcription factors in splenocytes 4 weeks after 2nd immunization will be measured by Luminex liquid phase chips and fluorescence quantitative PCR respectively. The influence of ECMS-oil on cytokines or chemokines production，kinetics and functional activity of lymphocytes at injection location or neighborhood lymph nodes after several inject time points will be measured by Luminex liquid phase chips method and flow cytometry respectively. The influences of ECMS-oil on phagocytosis activity from peritoneal macrophages, influences of ECMS-oil on the proliferation of lymphocytes and expression of related mRNA of cytokines or chemokines are also going to be measured. These results from the research will illuminate mechanism of the newly and efficient adjuvant.

波氏杆菌（Bb）是家兔呼吸道传染病重要病原之一，对养兔业危害严重。目前波氏杆菌疫苗免疫效果不十分理想，寻找安全高效疫苗佐剂将为规模兔场波氏杆菌引起的呼吸道感染防治提供可靠保障。本申请项目拟用ECMS-oil(木鳖子皂苷-白油)作为Bb灭活疫苗佐剂，利用酶联免疫吸附试验、淋巴细胞转化试验、Luminex液相芯片技术、荧光定量PCR、流式细胞术等技术，通过对ECMS-oil-Bb 免疫后总抗体及抗体亚类测定、2免后4周免疫兔淋巴细胞细胞因子和淋巴细胞中转录因子等表达变化测定；通过ECMS-oil对于兔天然免疫系统的免疫调节研究，包括研究在不同时间点ECMS-oil在局部注射后对注射局部微环境细胞因子或趋化因子产生影响，注射局部及邻近淋巴结组织免疫细胞动力学变化及功能状态变化的研究以及ECMS-oil对于兔腹腔巨噬细胞免疫调节作用的影响、ECMS-oil体外对兔脾淋巴细胞增殖和相关细胞因子转录因子mRNA的表达测定等内容阐明ECMS-oil对于Bb疫苗的佐剂作用及作用机理。

ECMS-oil对兔波氏杆菌疫苗佐剂作用研究结果表明ECMS-oil能显著提高兔波氏杆菌疫苗的体液免疫和细胞免疫效果。结果提示ECMS和油佐剂两者混合使用能够有效保护免疫兔。.ECMS-oil佐剂能显著提高小鼠抗波氏杆菌抗体水平。小鼠淋巴细胞增殖试验表明，EMCS-oil组能够显著提高小鼠T细胞免疫反应类型。小鼠脾脏mRNA相关基因荧光定量PCR分析结果发现小鼠ECMS-oil组的脾脏ASC、MyD88、STAT4、TLR2、TLR4、TLR6、TLR7等的表达显著上调，P<0.05。.ECMS-oil佐剂能显著提高家兔注射部位的白细胞、中间细胞和中性粒细胞。说明ECMS-oil佐剂能够比油佐剂更能激活淋巴细胞募集至注射部位，同时ECMS-oil在注射部位募集的淋巴细胞数量比油佐剂募集的更多，比如中间细胞和中性粒细胞还有白细胞。Luminex液相芯片结果表明ECMS-oil能下调IL-1a，IL-21，IL-17A，上调IL-1b，IL-8，MIP-1b，NCAM-1， MMP-9，TNA-a的分泌。.ECMS-oil组显著引起小鼠中性粒细胞、嗜酸性粒细胞、巨噬细胞的数量增加（P<0.01）,随时间推移，各组免疫细胞数量都呈下降趋势，且为显著性差异，P <0.05。ECMS在刺激Raw264.7后3小时的刺激指数显著高于对照组P<0.05。ECMS0.005μg刺激小鼠巨噬细胞的吞噬指数显著高于LPS组和ConA刺激组。ECMS-oil刺激下的小鼠脾淋巴细胞刺激指数显著高于其他各组。ECMS-oil能显著上调IL-4、 IL-10、 TLR2 、ASC-1表达，P<0.05。.Rg1-oil免疫试验结果说明Rg1-oil和oil能够显著提高免疫兔的体液免疫和细胞免疫水平。Rg1-oil能够显著提高免疫兔的细胞因子如IL-4、IFN-ɣ的表达水平。Rg1-Bb免疫兔外周血LncRNA测序结果。A3组对E组的上调基因为22个，下调基因为8个，B1组对E组的上调基因为16个，下调基因为8个，B2组对E组的上调基因为61个，下调基因为23个，B1组对B2组的上调基因为79个，下调基因为7个，B1组对A3组的上调基因为11个，下调基因为16个。Rg1-oil佐剂作用的机理与鞘糖脂生物合成-神经节系列，错配修复体，粘多糖的降解通路的调控相关。