IgG FcRn介导奶牛乳房炎无乳链球菌保护性抗原Sip亚单位疫苗的研究

Construction and clinical trial of the protective antigen Sip subunit vaccine against Streptococcus agalactiae in dairy cow mastitis mediated by IgG FcRn

为研究无乳链球菌Fc-Sip亚单位疫苗的免疫效果,并验证IgGFcRn在奶牛乳腺发生局部免疫过程中的重要作用。本研究采用大肠杆菌原核表达系统,表达了由IgGFcRn介导无乳链球菌保护性表面抗原蛋白(Sip)的融合蛋白Fc-Sip,并与佐剂混合制成Fc-Sip亚单位疫苗。选取干奶前15 d的奶牛进行加州乳房炎检测法(CMT)检测,选取120头CMT(++)以上的奶牛进行分组免疫。通过免疫前后的细菌分离试验、牛奶体细胞数测定、免疫牛血清抗体间接ELISA检测来评价该亚单位疫苗的免疫效果。结果显示,免疫前乳样细菌分菌率为86.6%,无乳链球菌检出率为46.6%;免疫后乳样分菌率降至40%,无乳链球菌检出率降至13.3%,表明Fc-Sip亚单位疫苗不但可以治疗无乳链球菌性奶牛乳房炎,还可以抑制其它奶牛乳房炎致病菌的侵入。首次免疫后60 d,免疫组80%奶牛血清抗体滴度达到1∶2 048～1∶4 096,对照组奶牛抗体滴度一直保持在1∶512。首次免疫后90 d,使用利拉伐牛奶体细胞计数仪测定实验奶牛乳样中的体细胞数,免疫组奶牛乳样体细胞数范围在0.17×10~5个/mL～3.91×10~5个/mL,对照组奶牛乳样体细胞数的范围在4.05×10~5个/mL～7.27×10~5个/mL。免疫组奶牛血清抗体滴度的升高及免疫后牛奶中体细胞数的减少,表明该亚单位疫苗促进了奶牛体液免疫反应和局部免疫反应的发生,也证明该亚单位疫苗具有降低牛奶体细胞数的作用。本研究为奶牛乳房炎无乳链球菌疫苗的研制奠定了基础。

In order to establish a platform for immunological study on the transport of specific antigen mediated by IgG FcRn in dairy cattle to prove. IgG FcRn plays an important role in mucosal immunity of bovine mammary glands. The fusion antigen gene encoding Fc-Sip of the Surface immunogenic protein （Sip） of cow IgG FcRn and Streptococcus agalactiae was constructed in this study. Then using Escolar prokaryotic expression system to express the Fc-Sip, and combined it with the adjuvant to prepare the subunit vaccine. A total of 120 CMT（＋＋） positive Holstein dairy cows at 15 days before the dry milk were selected for the immunization experiment. The effect of subunit vaccine was evaluated by bacteria isolation before and after immunization, determination of milk somatic cells and detection of the bovine serum antibody by indirect ELISA. The results showed that the total bacterial isolation rate was 86.6%, （Streptococcus lactis account for 46.6%） in the milk samples from the cows in experi- mental group before immunization, while, after immunization, the total of bacterial isolation rate decreased to 40% （S.lactis account for 13.3%） in the milk samples, Which indicated that the Fc-Sip immunization to cure the L.lactis mastitis, but also inhibited other dairy mastitis pathogenic bacteria invading. In addition, 60 days post the primary immunization, the bovine serum antibody titers reached 1：2,048 to 1：4,096 in 80% immunized cows, but the titers in the control group were all in 1：512. Moreover, 90 days post the initial immunization, the milk samples were detected by DeLaval milk somatic cell detector. The range of somatic cells in the majority of cows in the immunized groups were ranged from 0.17×10^5 to 3.91 ×10^5 cell/mL, while the somatic cells in the control group were around 4.05 ×10^5 to 7.27 ×10^5cell/mL. In conclusion, these data indicated that the Fe-Sip subunit vaccine was able to promote the occurrence of immune response in dairy cows, and it also proved that the subunit vaccine had the effect of reducing the number of somatic cells in milk of the S.agalactiae induced mastitis in dairy cow.