【作者】 苏良科；

【导师】 陆承平； 王永康；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 马链球菌兽疫亚种引致的猪链球菌病主要发生在我国，国外报道较少。该菌可引起牛、马的子宫内膜炎，流产，牛乳腺炎，猪的化脓性关节炎和败血症。它的致病因子包括非抗原性的透明质酸荚膜、透明质酸、链溶素O、链激酶、IgG Fc受体蛋白、肽聚糖和类M蛋白。在这些毒力因子中，最重要的是类M蛋白。它不但具有抗吞噬作用，而且还是链球菌良好的免疫原。所以它一直是研制链球菌疫苗的焦点。国内外迄今尚无理想的猪链球菌病疫苗。本试验的宗旨是从我国具体情况出发，研制安全高效、针对性强的马链球菌兽疫亚种类M蛋白的亚单位疫苗。 要研制疫苗，良好的免疫原是关键。为此，本文进行了以下方面的工作：首先用热酸法提取了马链球菌兽疫亚种中国株ATCC35246的类M蛋白，并克隆表达了ATCC35246类M蛋白位于胞外的、保守的抗原表位。再将热酸提取的类M蛋白、重组表达的类M蛋白和灭活的ATCC35246全菌分别免疫小鼠，通过对小鼠的保护率筛选出热酸提取的类M蛋白免疫原性最好。 亚单位疫苗由于仅含保护性免疫所需要的抗原，抗原表位不够丰富，免疫原性往往较弱，一般使用佐剂来弥补。为提高类M蛋白亚单位疫苗的免疫效果，将热酸提取的类M蛋白于铝胶、CpG和ISA206佐剂配合，免疫小鼠，拟筛选出免疫增强效果最好的一种佐剂。试验结果显示ISA206佐剂能激发快速长期的免疫反应，抗体效价高，而且对小鼠的刺激性弱，显示出该佐剂有望与类M蛋白亚单位疫苗配合，用于预防猪链球菌病。 为进一步了解类M蛋白的致病机制，本文以通用的HEp-2细胞为模型，研究了ATCC35246类M蛋白的粘附作用。试验结果表明，类M蛋白是ATCC35246株的粘附素之一，它可能是以N末端与HEp-2细胞表面受体结合；热酸提取的类M蛋白要比重组表达的类M蛋白对细菌粘附细胞的抑制作用要强，但两者都不能完全抑制粘附，显示链球菌还有其它有待发现的粘附机制存在。更多还原

【Abstract】 Streptococcus equi subsp. zooepidemicus causes disease in several animal species, including bovine mastitis, uterine infections and abortion of elderly horses, arthritis and septicimia of pigs. Its virulence factors include non-antigenic hyaluronic acid capsule, hyaluronidase. streptolysin O, streptokinase, IgG Fc receptor proteins, peptideglycan and M-like protein. M-like protein, an a -helical coiled-coil molecule attached to the cell membrane and extending out the capsule, elicits very strong B and T cell responses resulting in protective immunity mediated by opsonic antibodies. However, vaccines based on acid or mutanolysin extracted M-like protein mainly focused rather on the prevention of strangles of foals than on that of swine streptococcosis. To develope a highly efficient and safe M-like protein subunit vaccine against S. zooepidemicus infection of swine is expected.Hot-acid extracted M-like protein of S. zooepidemicus strain ATCC35246, the recombinant M-like protein and the whole inactive ATCC35246 cells were immunized mice respectively with ISA206 adjuvant to select the optimal antigen. The challenge test showed that hot-acid extracted M-like protein had quite strong antigenicity and the potential value to be developed as subunit vaccine.Subunit vaccines often induce weak immune response and protection when administered alone. In order to improve the efficacy of M-like protein subunit vaccine, three adjuvants, aluminium hydroxide, bacterial CpG and ISA206 were tested. They were injected into mice together with hot-acid extracted M-like protein separately. The results showed that ISA206 adjuvant could induce short and long term immunity without obviously adverse reactions, and its immune enhancement was higher than that of the other two. It suggested that ISA206 adjuvant could be used as an immunoadjuvant for the prevention of swine streptococcosis.In addition, adhesion role of M-like protein from S. zooepidemicus strain ATCC35246 to HEp-2 cells was evaluated by the adhesion and adhesive inhibition experiments. Evidence presented in the study indicated that M-like protein was a component of the adhesion mechanism of S. zooepidemicus strain ATCC35246, andthe binding site might reside near the N terminus. The inability of hot-acid extracted and recombinant M-like protein to completely inhibit streptococcal adhesion was probably due to alternative adhesions.更多还原