羊布氏菌P39和L7/L12基因重组BCG的构建及其免疫机制研究

Brucellosis is a zoonosis caused by bacteria of the genus Brucella. Inner Mongolia is the most serious area sufferring from Brucellosis in China, and Brucella melitensis has been confirmed to be the most important agent. Brucella spp. is a Gram-negative, facultative intracellular bacterium, therefor, the humoral and cell-mediated immune responses are both needed in controlling the disease including B lymphocyte, CD4+ and CD8+ T lymphocytes, Th1-type cytokines such as inerferon-gamma (IFN-γ) and tumor necrosis factor (TNF-α), and activated macrophages and dendritic cells. Despite the availability of live vaccine strains, the vaccines have several drawbacks, including interference with diagnosis, resistance to antibiotics, and residual virulence, that prevent their use in humans. Previous researches have indentified Brucella genes coding periplasmic binding protein, outer membrane proteins and metabolic enzymes. Among them are P39 and L7/L12 genes which encode the 39kDa periplasmic binding protein (PBP39) and ribosomal protein L7/L12 respectively. And the PBP39 and L7/L12 are considered important immunogens in Brucella melitensis. The Bacille Calmette Guerin (BCG) is the current vaccine for tuberculosis, also could be used as an immunologic adjuvant and a desired host expressing foreign antigen genes. The study is planned to construct a recombinant BCG (rBCG) expressing the antigen coding genes (P39 and L7/L12 genes) from Brucella melitensis. The immune responses of mice will be systematically detected immunized with rBCG and INF-γ as an adjuvant and the immune mechanism of the rBCG will also be inspected. Furthermore, the project is designed to demonstrate the feasibility of expressing dominant antigens coding genes from Brucella in BCG.

布鲁氏菌病是由布氏菌感染引起的一种人兽共患传染病。内蒙地区布病发病率连年位居全国之首，羊布氏菌为主要流行株。布氏菌是细胞内寄生菌，同时诱导机体产生体液免疫和细胞免疫应答才能有效地预防与控制该病的流行和传播。现有疫苗毒性较大且不能区别自然感染与免疫接种，限制了其在人群中的使用。羊布氏菌周质结合蛋白P39和核糖体蛋白L7/L12具有很强的免疫原性，可诱导机体产生特异性免疫应答。IFN-γ具有广泛的免疫调节作用，可以显著提高抗原的免疫效应。卡介苗（BCG）既是防治结核病的减毒疫苗，又具有明显的免疫佐剂效应，可有效增强细胞免疫功能，同时也是一种理想的抗原表达载体。本研究拟构建携带羊布氏菌P39和L7/L12基因的重组BCG并以IFN-γ为佐剂，以期诱导产生有效的免疫应答，对该重组BCG的免疫效果和免疫机制进行系统的研究，为探索利用重组BCG表达布氏菌优势抗原的可行性及其免疫机制奠定可靠的实验基础。

布鲁氏菌病是由布氏菌感染引起的一种人兽共患传染病。内蒙地区布病发病率连年位居全国之首，羊布氏菌为主要流行株。现有的布病减毒疫苗存在着毒性大且不能区别自然感染与免疫接种等诸多问题，限制了其在人群中的使用。新型布病疫苗一直是布病研究领域的热点问题。本项目利用大肠杆菌-结核分枝杆菌穿梭表达载体（pMV361），构建了3种携带不同布氏菌优势抗原编码基因的重组表达质粒，并将其转染卡介苗（BCG）后获得相应的重组BCG疫苗。实验结果表明，参照BCG基因组进行密码子优化和温度诱导（60℃诱导4小时），布氏菌P39基因、L7/L12基因和P39-L7/L12融合基因在BCG中的表达水平明显提高。携带布氏菌P39和L7/L12融合基因的重组BCG（rBCG-P39-L7/L12）免疫BALB/c小鼠4周后，能够诱导产生特异性anti-P39和anti-L7/L12抗体；相比现有的M5株疫苗，rBCG-P39-L7/L12疫苗能够促进实验小鼠产生更多的IL-12、IFN-γ、TNF-α和IL-10等细胞因子，提高外周血中CD3+CD8+T细胞比例（与对照组相比）；以IFN-γ为佐剂的rBCG-P39-L7/L12疫苗方案则有利于IL-2的表达，增强脾脏NK细胞非特异性杀伤功能，提高外周血中CD3+CD4+T细胞和CD3+CD8+T细胞比例（与对照组相比）。3组疫苗组外周血Treg细胞和脾脏Th17细胞比例均升高。初步攻毒试验结果表明，M5株疫苗能够赋予实验小鼠良好的保护作用；rBCG-P39-L7/L12疫苗和以IFN-γ为佐剂的rBCG-P39-L7/L12疫苗都能够为实验小鼠提供一定的保护作用，但两种之间的差异不具有统计学意义。本研究在利用BCG为表达宿主进行新型布病疫苗研究方面进行了有意义的尝试和探索，也为后续的研究奠定良好的基础。