重组大肠杆菌不耐热肠毒素的表达及对小鼠胚胎稳定性的影响

为研究大肠杆菌不耐热肠毒素（LT）对小鼠胚胎稳定性的影响,运用大肠杆菌表达系统制备了具有生物活性的重组大肠杆菌LT,用重组LT通过腹腔注射处理妊娠小鼠,分析了LT对小鼠胚胎稳定性的影响。首先采用PCR方法从产毒大肠杆菌菌株44815基因组中扩增LT的A、B亚基基因,将其插入到pET-20b（＋）原核表达载体pelB信号肽的下游,分别构建成LTA和LTB分泌型表达载体;将载体分别转化大肠杆菌BL21（DE3）pLysS,在IPTG的诱导下进行表达,利用Ni-NTA琼脂糖凝胶从细菌周质释放液中提取和纯化重组LTA和LTB蛋白;利用细胞毒性试验检测重组蛋白的生物活性。然后用制备的重组LT注射妊娠6d的小鼠,连续注射3d后,统计小鼠的胚胎存活率。同时用ELISA方法检测小鼠血清中与胚胎稳定性密切相关的Th1型细胞因子（IFN-γ、IL-2）和Th2型细胞因子（IL-4、IL-10）及IL-β的表达水平。结果表明,采用分泌性表达策略实现了重组LT在大肠杆菌中的高效分泌表达,LTA和LTB的表达量分别达68、62mg/L。表达的重组LT具有明显的细胞毒性作用;用LT处理妊娠小鼠,胚胎的存活率为32%,明显低于对照组;小鼠血清中Th1型细胞因子IFN-γ和IL-2的含量明显高于对照组（P〈0.01）,分别为对照组的2、3倍。同时细胞因子IL-1β为对照组的2倍（P〈0.01）,而稳定胚胎发育的Th2型细胞因子IL-4、IL-10没有明显变化（P〉0.05）。据此推测,LT可能与大肠杆菌性肠炎引起妊娠家畜流产有关,LT对胚胎稳定性的影响除与LT的直接毒性有关外,可能还与LT介导的免疫调节异常有关。

Expression of recombinant E. coli heat-labile enterotoxin and its effects on stability of mouse embryo

To characterize the effects of E. coli heat-labile enterotoxin （LT） on stability of mouse embryo, we ex pressed LT using E. coli expression system,and analyzed its effects on mouse embryo stability by the intraperitoneal injection with recombinant LT. Firstly, the A and B subunits of LT gene were amplified from genomic DNA of enter- otoxigenic E. coli strain 44815. The LTA and LTB genes were inserted into the downstream of pelB signal peptide sequence in pET-20b（＋） vector to construct LTA and LTB secretory expression vectors, respectively. The vectors were transformed into E. coli BL21（DE3） pLysS for LTA and LTB expression under the induction of IPTG. The LT proteins were purified with Ni-NTA agarose beads and were analyzed for the biological activity by the cytotoxicity assay. The pregnant mice were treated with LT at the 6th day of pregnancy. After 3-day continuous treatment, the embryo survival rates were figured,the Th1 type cytokines （IFN-γ,IL-2）,Th2 type cytokines （IL-4, IL-10） and IL- 1β were detected by ELISA. The results showed that by adopting secretary expression strategy, the high-level LT expression was achieved in E. coli,the yields of LTA and LTB were 68 mg/L and 62 mg/L,respectively. The recombi- nant LT showed obvious eytotoxicity. The embryo survival rate of LT-treated pregnant mice was 32 %,significantly lower than that of the untreated mice. The concentrations of Thl type cytokines （IFN-7, IL-2） in the serum of the LT-treated mice increased by 2-fold and 3-fold （P〈0.01） compared with the control mice, respectively. And the IL- 1β also increased by 2-fold （P〈0.01） compared with the control group. However, the Th2-type cytokines （IL-4, IL- 10） were no significant change（P〉0.05）. These results indicate that the LT has the effects on mouse embryo stability,which was supposed to be closely related not only with the LT cytotoxicity,but also with the immunoregulation of LT.