异烟肼处理前后耻垢分枝杆菌mc^2 155细胞蛋白双向电泳图谱的差异分析

目的利用蛋白质组学方法,识别异烟肼处理前后耻垢分枝杆菌mc2 155细胞(SM细胞)后差异表达的蛋白质,为进一步深入探讨异烟肼抑制分枝杆菌生长的作用机制及寻找新的抗结核药物作用靶标奠定基础。方法观察不同浓度的异烟肼处理前后耻垢分枝杆菌生长曲线,确定药物处理SM细胞的最佳时间和浓度,并提取在最佳处理时间和浓度条件下的SM细胞总蛋白,采用载体两性电解质pH梯度双向凝胶电泳技术分离异烟肼处理前后耻垢分枝杆菌mc2 155细胞的总蛋白质,用PDuest7.3.1图像分析软件比较分析,以识别差异表达的蛋白质。结果在空白对照组检出159个蛋白斑点,在异烟肼处理组检出221个蛋白斑点,在相对分子质量30 000~90 000和pH值4.5~6.0范围内,异烟肼处理组蛋白斑点数均多于空白对照组。结论异烟肼处理前后耻垢分枝杆菌mc2155细胞的蛋白质组具有差异,提示异烟肼处理后促进了某些特定基因的表达。

表达人幽门螺杆菌外膜蛋白的穿梭质粒的构建及在耻垢分枝杆菌中的表达

目的建立表达人幽门螺杆菌外膜蛋白重组耻垢分枝杆菌疫苗株,为进一步应用重组耻垢分枝杆菌疫苗防治幽门螺杆菌感染打下基础。方法利用聚合酶链反应(Polymerasechainreaction,PCR)扩增的幽门螺杆菌的外膜蛋白528编码基因全长片段,克隆入pET32a(+),鉴定无误后,再亚克隆入大肠杆菌-分枝杆菌穿梭质粒pLA71,构建pLA71-omp载体,将pLA71-omp电转化耻垢分枝杆菌,经卡那霉素筛选后,抽提质粒用PCR法鉴定,Westernblot检测omp的表达及活性。结果从幽门螺杆菌基因组中扩增出约528bp的基因片段。所构建重组体阳性克隆经酶切和PCR鉴定与预期结果一致,测序结果确证了插入片段的正确性,Westernblot检测说明幽门螺杆菌的外膜蛋白528编码基因在分枝杆菌中获得了表达并具有良好的免疫原性。结论成功构建了幽门螺杆菌外膜蛋白528编码基因大肠杆菌-分枝杆菌穿梭表达质粒。该质粒能在E.coli/MS间进行穿梭,并在耻垢分枝杆菌中表达。

Comparative Study on the Immunogenicity between Recombinant MS-Sj26GST Vaccine and Recombinant BCG-Sj26GST Vaccine in Schistosoma japonicum

The BALB/c mice were immunized with rMS Sj26GST and rBCG Sj26GST vaccine in Schistosoma japonicum by subcutaneous injection After they were immunized for 8 weeks, the eyeballs were removed to get blood and macrophages of abdominal cavity and spleen cells were harvested The lymphocytic stimulating index (SI) was used to measure the cellular proliferating ability and NO release was used to measure the phagocytic activity of the macrophages By using ELISA kit, the levels of interleukin 2 (IL 2) and interferon γ (IFN γ) in serum and the splenic lymphocytic cultured supernatant were detected The results showed that after the mice were immunized with 10 6 CFU of rMS Sj26GST and rBCG Sj26GST vaccine separately by subcutaneous injection, proliferating ability of splenic lymphocytes in the mice showed no difference ( P >0 05), but both were significantly increased as compared with that in the control group( P <0 05); The contents of NO in the intraperitoneal macrophages of rMS Sj26GST vaccine group were significantly lower than in the control group ( P <0 001) and rBCG Sj26GST vaccine group ( P <0 01); The levels of serum IL 2 in the rMS Sj26GST vaccine group were significantly increased as compared with that in the control group ( P <0 001), vector group ( P <0 01) and rBCG Sj26GST vaccine group ( P <0 05); The contents of serum IFN γ in the rMS Sj26GST vaccine group were significantly increased as compared with that in the control group ( P <0 01) and rBCG Sj26GST vaccine group ( P <0 05) The contents of IFN γ in the cultured supernatant were significantly lower than those of rBCG Sj26GST vaccine group ( P <0 001), but were significantly increased as compared with that in the control group ( P <0 01) It was indicated that both vaccines could enhance the immune response of the mice, but rMS Sj26GST vaccine had stronger immunogenicity than rBCG Sj26GST vaccine

构建CRISPR-Cas9介导的耻垢分枝杆菌基因组高效删除系统

【背景】耻垢分枝杆菌具有生长迅速和非致病性的特点,可作为结核分枝杆菌致病机理研究替代菌株和类固醇激素生产的工程菌,但目前耻垢分枝杆菌中缺乏高效率的基因组敲除方法。【目的】基于CRISPR-Cas9介导的定点、高效的DNA切割能力,构建耻垢分枝杆菌染色体DNA片段无痕敲除系统。【方法】构建了包含四环素诱导型启动子驱动的密码子优化的cas9基础载体pCas9101,在双侧同源臂长度约为1 kb条件下选用合适的gRNA表达模块,分别测试了对耻垢分枝杆菌mc2155染色体上的3β-羟基类固醇脱氢酶基因(MSMEG_5228,1 071 bp)和胆固醇降解基因簇(MSMEG_5990-MSMEG_6043,约48kb)敲除效率,使用相同大小的同源臂以经典p2NIL-pGOAL方法进行对照,并计算效率。【结果】使用CRISPR-Cas9方法对耻垢分枝杆菌mc2155的3β-羟基类固醇脱氢酶基因敲除效率为22%,胆固醇降解基因簇敲除效率也达到18%,两者连续敲除效率为4%。但对照p2NIL-pGOAL方法未能获得目标DNA片段敲除的菌株。【结论】本文建立的基于CRISPR-Cas9的耻垢分枝杆菌基因组无痕敲除系统显示出较高的敲除效率,该方法可为耻垢分枝杆菌后续研究提供快速高效的基因组操作方法。