产毒大肠杆菌肠毒素质粒(Entg::Tn5)的转导实验研究

Study on Transduction of Ent::Tn5 Plasmid Harboring in Escherichia Coli K_(12)W_(1485)

我们利用1株带有Km抗性基因的产毒大肠杆菌E·colikl2W1485(Ent::Tn5)作为P1增殖菌制备P1(LT)裂解液,11株分属E、coli、E1EC、S.dysentery、S、sonnei、S.boydii、Proteus菌株作为受体菌,进行试管内转导试验。结果表明11株受体菌中有9株发生质粒完全和部分转导,分别呈表型K_m^+LT^+和K_m^+LT^-。选c600/P1(LT)转导子进行质粒电泳检测,发现K_m^+LT^+转导子出现同P1(LT)增殖菌E、colikl2相同的质粒带,而K_m^+LT^-未出现质粒带。我们再从11株受体菌中选用7株进行兔肠内转导实验,结果发现有3株受体菌所在肠段明显肿胀;而另4株虽不能引起肠段肿胀,但也可分离出转导子。同时,选用10株受体菌进行小白鼠灌胃质粒转导实验,其中4组可分离出转导子。 试管内转导子保留半年后,除1株K_m^+LT^+转导子失去质粒外,其它均保留其原表型。说明转导子稳定性良好。据上述结果提示Ent质粒和R质粒可通过转导在自然界传播。

P1 (LT) lysate, prepared by infecting E. coli K 12 W 1485 (Ent::Tn5) with PI lysate, were used to infect 11 strains of E. coli, EIEC, S. dysentery, S. sonnei, S. boydii and Proteus in vitro. As a result, partial or complete transduction occurred in 9 strains, which showed pheno-type of Km+LT- or Km4LT+. 4 strains of transductant C600/P1 (LT) were examined by a-garose gel electrophoresis. It was found that Km+LT + had the same plasmid zone as E. coli W 1485 (Ent::Tn5) while Km+LT- did not present any plasmid zone. Out of the 11 strains 7 were chosen to conduct transduction inside rabbit intestines. The result showed that intestinal parts where 3 strains were injected respectively, together with PI (LT, were swollen evidently. Though the intestinal parts injected with P1 (LT) other 4 strains, were not swollen, transduc-tants were got from the intestinal parts. Simultaneously,transduction experiments were conducted inside white mice's stomach and the transductants were also isolated.After being kept in room temperature for six months, the transductants getting from transduction experiments in vitro still retained their phenotype except one transductants losing its phenotype Km4LT4 and also its plasmid zone.We infer that Ent and R plasmids may spread in nature by transduction.