【摘要】 目的检测先天性弓形虫感染胎鼠脑组织差异蛋白并分析其功能,为先天性弓形虫病的发生机制研究提供理论基础。方法选取9~10周龄BALB/c孕鼠20只,随机分为正常对照组和感染组,每组10只。于妊娠第10d,感染组每只小鼠腹腔注射经胰酶消化的弓形虫速殖子40个/0.2ml;正常对照组腹腔注射无菌PBS溶液0.2ml/只(0.01mol/L,pH 7.3)。于妊娠第20d处死孕鼠,取胎鼠脑组织,采用Bradford法测定蛋白含量;采用双向凝胶电泳分离胎鼠脑组织蛋白,运用ImageMaster 2D软件分析差异蛋白,采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)分析差异蛋白点的肽指纹谱(PMF),运用Mascot软件在SWISS-PROT数据库中对差异蛋白进行比较与评判,运用David软件对差异蛋白进行生物信息学分析。结果两组胎鼠脑组织均分离出约500个蛋白点,经蛋白质胶图分析鉴定出显著差异蛋白点15个,分子质量单位(Mr)多分布于(8~48)×103之间,等电点(pI)在3~10之间。在弓形虫感染胎鼠脑组织低表达的15个差异蛋白点经过鉴定和功能分析,主要是以下6种:结蛋白,肌动蛋白,血清白蛋白,葡萄糖调节蛋白,蛋白质二硫键异构酶和膜联蛋白。结论弓形虫感染胎鼠脑组织中有6种差异蛋白低表达,弓形虫可能通过降低这些蛋白的表达量来影响胎鼠脑组织的正常结构、物质转运和调节功能等,从而引发先天性弓形虫脑病。更多还原

【Abstract】 Objectives To detect differences in proteins in the brain of fetal mice with a congenital Toxoplasma gondii infection and to analyze their functions in order to provide a theoretical basis for the study of the mechanism of congenital toxoplasmosis. Methods Twenty 9-10-week-old BALB/c pregnant mice were randomly divided into a control group and an infection group.On the 10 th day of pregnancy,each mouse in the infection group was intraperitoneally injected with 40 trypsin-digested tachyzoites/0.2 ml;pregnant mice in the control group were intraperitoneally injected with 0.2 ml of a sterile PBS solution/mouse(0.01 mol/L,pH 7.3).On the 20 th day of pregnancy,pregnant mice were sacrificed,and fetal brain tissue was collected from each group.The protein content in fetal brain tissue was determined using the Bradford method.The fetal brain proteins were separated using two-dimensional gel electrophoresis(2-DE).Differences in proteins were analyzed using the software ImageMaster 2 D.The peptide mass fingerprinting(PMF)of different protein spots was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS).Differences in proteins were compared and evaluated with the SWISS-PROT database using the software Mascot.The software David was used to bioinformatically analyze differences in proteins. Results About 500 protein spots were produced by fetal brain tissue from the two groups.Fifteen protein spots were identified using protein gel analysis.The molecular mass(Mr)of the proteins mainly ranged from 8-48× 10~3,and the isoelectric point(pI)ranged from 3-10.The 15 fetal brain proteins that were expressed in lower levels in animals with a T.gondii infection were identified and functionally analyzed.These proteins mainly consisted of 6 proteins:desmin,actin,serum albumin,glucose-regulated protein,protein disulfide isomerase,and annexin. Conclusion Six proteins were expressed at lower levels in the brain tissue of fetal mice infected with T.gondii.T.gondii may reduce the expression of these proteins and thus affect the normal structure of fetal brain tissue,substance transport,and regulatory functions.This may account for the encephalopathy noted in congenital toxoplasmosis.更多还原