田鼠巴贝虫肽基脯氨酸异构酶重组蛋白免疫保护效果

Immunoprotective effect of recombinant peptidyl-prolyl cis-trans isomerase from Babesia microti against B.microti infection in mice

目的观察田鼠巴贝虫肽基脯氨酸异构酶(BmPPIase)重组蛋白主动免疫对巴贝虫感染小鼠的免疫保护效果。方法将雌性BALB/c小鼠(6周龄,约20 g/只)分为重组蛋白免疫组、感染对照组、正常对照组,各组分别为25、18、15只。以BmPPIase重组蛋白主动免疫重组蛋白免疫组小鼠,末次免疫后2周,选取抗体效价较高的18只小鼠经腹腔注射100μL田鼠巴贝虫染虫全血;感染对照组小鼠均经腹腔注射100μL田鼠巴贝虫染虫全血;正常对照组15只小鼠不进行任何处理,直接进行后续实验。重组蛋白免疫组、感染对照组于实验第0~30天,采血观察各组小鼠巴贝虫感染情况,计算染虫率;此外,于实验第0、7、14、21、28天采用血细胞分析仪对各组小鼠进行血常规检测,并采用微量样本多指标流式蛋白定量技术进行小鼠血清细胞因子检测。结果末次免疫后2周,重组蛋白免疫组25只小鼠体内均产生抗BmPPIase抗体,效价为5×10^(3)~8×10^(4)。重组蛋白免疫组和感染对照组小鼠均于实验第7天达到染虫高峰,染虫率分别达13.3%和50.0%;两组染虫率在实验第3、5、7、9天差异均有统计学意义(χ^(2)=113.18、475.22、465.98、18.71,P均<0.01);实验第11天后,两组染虫率均逐渐趋于0。全血细胞分析显示,感染第0~28天,重组蛋白免疫组小鼠红细胞数量[(5.30±0.50)×10^(12)/L~(9.87±0.24)×10^(12)/L]及血红蛋白含量[(89.67±22.80)~(148.60±3.05)g/L]均高于感染对照组。观察期间内重组蛋白免疫组小鼠血清γ干扰素(IFN-γ)[(748.59±17.56)~(3858.28±1049.10)fg/mL]、肿瘤坏死因子α(TNF-α)[(6687.34±1016.64)~(12708.13±1629.79)fg/mL]、白细胞介素(IL)-6[(611.05±75.60)~(6852.68±1554.00)fg/mL]及IL-17a[(167.68±185.00)~(10849.27±355.40)fg/mL]均高于感染对照组,而IL-10[(247.65±138.00)~(18787.20±2830.22)fg/mL]低于感染对照组。结论田鼠巴贝虫PPIase重组蛋白可诱导感染小鼠体内IFN-γ、TNF-α等重要细胞因子上调表达,对宿主有较好的免疫保护效果,为一种极具潜力的疫苗候选蛋白。

Objective To evaluate the immunoprotective effect of active immunization with recombinant peptidyl-prolyl cis-trans isomerase from Babesia microti against B.microti infection in mice.Methods Female BALB/c mice at 6 weeks of age,each weighing approximately 20 g,were divided into the recombinant protein immunization group,the infection control group and the normal control group,of 25,18,15 mice in each group,respectively.Mice in the recombinant protein immunization group were given active immunization with recombinant BmPPIase protein,and 18 mice with the highest antibody titers were intraperi-toneally injected with 100μL of B.microti-infected whole blood 2 weeks after the last immunization.Mice in the infection control group were intraperitoneally injected with 100μL of B.microti-infected whole blood,while 15 mice in the normal control group received no treatment.Blood samples were collected from mice in the recombinant protein immunization group and the infection control group on days 0 to 30 post-immunization for detection of B.microti infection,and blood samples were collected on days 0,7,14,21,and 28 post-immunization for routine blood tests with a blood cell analyzer and for detection of serum cytokines using cytometric bead array.Results Anti-BmPPIase antibodies were detected in 25 mice in the recombinant protein immunization group 2 weeks after the last immunization,with titers of 5×10^(3)to 8×10^(4).B.microti infection rate peaked in mice in both the recombinant protein immunization and the infection control group on day 7 post-immunization,with positive infection rates of 13.3% and 50.0%,and there were significant differences between the two groups in terms of B.microti infection rate on days 3(χ^(2)=113.18,P<0.01),5(χ^(2)=475.22,P<0.01),7(χ^(2)=465.98,P<0.01)and 9 post-infection(χ^(2)=18.71,P<0.01),while the B.microti infection rate tended to be 0 in both groups on day 11 post-immunization.Routine blood tests showed higher red blood cell counts[(5.30±0.50)×10^(12)to(9.87±0.24)×10^(12)counts/L]and hemoglobin levels[(89.67±22.80)to(148.60±3.05)g/L]in the recombinant protein immunization group than in the infection control group on days 0 to 28 post-immuniza-tion.Cytometric bead array detected higher serum interferon-γ[(748.59±17.56)to(3858.28±1049.10)fg/mL],tumor necrosis factor-α[(6687.34±1016.64)to(12708.13±1629.79)fg/mL],interleukin(IL)-6[(611.05±75.60)to(6852.68±1554.00)fg/mL]and IL-17a[(167.68±185.00)to(10849.27±355.40)fg/mL]and lower IL-10 levels[(247.65±138.00)to(18787.20±2830.22)fg/mL]in the recombinant protein immunization group than in the infection control group during the study period.Conclusions Recombinant BmPPIase protein induces up-regulation of interferon-γ,tumor necrosis factor-αand presents a high im-munoprotective activity against B.microti infection in mice,which is a potential vaccine candidate protein.