表达蝎毒素AaIT或苏云金杆菌毒素Cyt2Ba的大肠埃希菌杀蚊幼活性及增效剂型测试

发布时间：2018-04-27 14:34

  本文选题：AaIT + CytBa　； 参考：《南方医科大学学报》2017年06期

【摘要】：目的 构建表达蝎毒素AaIT或苏云金杆菌毒素Cyt2Ba的大肠埃希菌,检测重组菌对致倦库蚊和白纹伊蚊II龄幼虫的毒力,并测试不同剂型的增效作用。方法 分别将AaIT和Cyt2Ba编码基因克隆于pET28a(+)质粒上,并将重组质粒分别转化BL21 E.coli获得分别表达AaIT和Cyt2Ba的工程菌株,经IPTG诱导后,用SDS-PAGE和Western-blot检测重组蛋白的表达;测试不同浓度工程菌菌液对于蚊幼虫的杀灭效果;并将有效的工程菌制成干粉剂,检测其干粉末及其配剂对蚊幼虫的杀灭效果。结果 AaIT和Cyt2Ba重组蛋白在大肠埃希菌中成功表达,但AaIT重组蛋白对蚊幼虫不具有毒性。表达Cyt2Ba重组蛋白的工程菌菌液对白纹伊蚊和致倦库蚊幼虫均具有明显的杀灭作用,48 h的LC50分别为3.00×106和1.25×107cells/m L,其中对白纹伊蚊幼虫的杀灭效果要显著优于对致倦库蚊幼虫的杀灭效果(P0.001),并且表达Cyt2Ba重组蛋白的工程菌干粉末及其配剂均具有较好的杀蚊幼虫效果。当该工程菌干粉末与干酵母粉、小麦粉和白胡椒粉分别按4∶1、1∶1和1∶4比例制成配剂处理蚊幼虫48 h后,结果显示当质量比为1:1时,配剂杀蚊幼效果显著提高(P=0.044),并且白胡椒粉末配剂效果要显著好于干酵母粉和小麦粉配剂(P=0.002)。结论 表达Cyt2Ba的重组大肠埃希菌对蚊幼虫具有较好的杀灭作用,合适配剂的使用可以增强其在蚊虫防制中的效果。
[Abstract]:Objective to construct Escherichia coli expressing scorpion toxin AaIT or Bacillus thuringiensis Cyt2Ba to detect the virulence of recombinant bacteria against Culex pipiens quinquefasciatus and Aedes albopictus II instar larvae and to test the synergism of different dosage forms. Methods the AaIT and Cyt2Ba coding genes were cloned into pET28a () plasmid, and the recombinant plasmid was transformed into BL21 E.coli to obtain the engineering strain expressing AaIT and Cyt2Ba respectively. After IPTG induction, the expression of recombinant protein was detected by SDS-PAGE and Western-blot. The killing effect of different concentrations of engineering bacteria liquid on mosquito larvae was tested, and the effective engineering bacteria were made into dry powder to detect the killing effect of dry powder and its mixture on mosquito larvae. Results AaIT and Cyt2Ba recombinant proteins were successfully expressed in Escherichia coli, but AaIT recombinant proteins were not toxic to mosquito larvae. The LC50 of the engineering liquid expressing Cyt2Ba recombinant protein against both Aedes albopictus and Culex pipiens quinquefasciatus larvae was 3.00 脳 10 ~ 6 and 1.25 脳 107cells/m / L for 48 h, respectively. The killing effect on Aedes albopictus larvae was significantly better than that on Culex pipiens quinquefasciatus larvae. The insecticidal effect of larva was P0.001, and the dry powder and its mixture of recombinant Cyt2Ba protein had better effect on killing mosquito larvae. When the dry powder, dry yeast powder, wheat flour and white pepper powder were made into a mixture of 4: 1: 1: 1 and 1:4 to treat mosquito larvae for 48 h, the results showed that when the mass ratio was 1:1, The effect of the mixture on mosquito control was significantly improved, and the effect of white pepper powder was better than that of dry yeast powder and wheat flour mixture (P0. 002). Conclusion Recombinant Escherichia coli expressing Cyt2Ba has a good killing effect on mosquito larvae, and its effect on mosquito control can be enhanced by the use of suitable compound.
【作者单位】： 南方医科大学公共卫生学院病原生物学系//广东省热带病研究重点实验室;
【分类号】：R184.31

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