奶牛乳腺炎Ia型无乳链球菌菌毛岛屿PI-2a多亚基不同组合融合蛋白抗原性研究

The pili of dairy bovine mastitis Streptococcus agalactiae play an key role in adhesion to host cells,and was also the important adhesion fector and candidate immune target.Pili was the three subunit structure that which was composed of subunit BP,AP1,AP2 under the action by sortase.Pilus subunit proteins of GBS from humman showed a certain degree of protective immunity to mouse,however,the immunologic mechanism of the pilius of GBS from diary cow subunit fusion antigen to mouse was unclear.In prospective study, we have found that the 90 percent of isolated bacterial strains were Ia serotype.And 91 percent of them contained PI-2a pilus island.We have confirmed that AP1 subunit protein of PI-2a showed good antigenicity.Accordingly,we proposed to construct multi-subunit antigens of PI-2a to study the immune characteristics in dairy bovine.We will clone the coding sequences of the three genes of BP,AP1,AP2 by the PCR and will construct the fusion genes baa by repeat PCR.The fusion genes will be inserted into expression vector pET, and after will be identificatifyed and sequenced to be transfered into enginereing E. coli BL21 and to be induced and expressed.The expressed fusion protein will be purified and to be used with immune to mouse and to product antibody.After the growth inhibition effect of bacterial strain will be analyzed by FACS and the adherence inhibition effect identificated by bovine mammary gland cellular layer,the immunization experiments in Babl/c mouse will be implemented with the purified fusion antigens.The objective of this study will to explore the immune protective effect and the antibodise law and which will have an important scientific significances for to estimate the immune mechanism of the BP,AP1 and AP2 multi-subunit fusion antigens in Balb/c mouse.This study will provide a new idea for further study the protection and control new ways and new type of immune antigens of dairy bovine mastitis.

牛乳腺炎无乳链球菌菌毛在其黏附宿主细胞过程中起到关键作用,是重要的粘附因子和免疫侯选靶标，是由BP、AP1及AP2亚基聚合形成的三亚基结构。人源无乳链球菌菌毛PI-2a亚基对小鼠表现良好的免疫保护性,但是牛乳腺炎无乳链球菌菌毛PI-2a亚基因序列因有差异，从而其亚基蛋白对小鼠能否具有免疫保护作用并不清楚。已发现内蒙古分离株90%存在PI-2a菌毛基因，同时证实AP1亚基具有良好的抗原性，据此提出构建PI-2a菌毛岛屿亚基不同组合融合抗原对Balb/c小鼠进行免疫实验，预期明确其抗原性的设想。将用PCR技术构建PI-2a菌毛岛屿BP、AP1、AP2亚基序列不同组合融合基因，再构建重组工程菌获得不同组合融合抗原。经体外流式细胞仪分析对菌株生长抑制效果及对牛乳腺细胞爬片黏附抑制作用等免疫反应性基础上，对小鼠进行免疫保护实验，以期筛选最佳亚基组合抗原，为丰富牛乳腺炎防控提供新的科学理论依据。

奶牛乳腺炎是奶牛的重要疫病之一，由无乳链球菌引起的奶牛乳腺炎在内蒙古东部区多发，其病原无乳链球菌是重要的人畜共患致病菌之一，临床乳腺炎乳样中检出率达到70%以上，除了造成牛乳减产、奶牛淘汰等巨大经济损失外，还具有重要的乳源安全及公共卫生意义。本项目从该致病菌重要致病因子PI-2a菌毛岛屿基因入手，克隆并获得AP1、BP、AP2三个亚基基因不同组合基因片段AP1-BP、BP-AP2、AP1-AP2、AP1-BP-AP2及其表达蛋白抗原，对其抗原性通过免疫家兔及western-blot鉴定后，明确均有不同程度的抗原性的基础上，最终对小鼠进行了不同组合抗原的免疫保护实验。结果显示，对实验小鼠的免疫保护率指数PI达到80%，对科尔沁奶牛乳区免疫保护率指数达到80.5%，达到了预期的研究目标，获得了内蒙古地区奶牛乳腺炎临床分离Ia型无乳链球菌PI-2a菌毛岛屿AP1-BP-AP2亚基重组融合抗原对小鼠和奶牛具有很好的免疫保护效果的科学结论，丰富了奶牛乳腺炎防控理论，为进一步研究亚单位重组基因工程疫苗及快速检测乳腺炎试剂盒奠定了坚实的研究基础。