摘要
目的 克隆表达肠出血性大肠杆菌（EHEC）O157：H7志贺样毒素Ⅱ（ShigaliketoxinⅡ,Stx2）,并鉴定其生物学活性.方法 采用PCR法自EHEC O157：H7基因组扩增志贺样毒素Ⅱ的编码基因stx2,T-A克隆后构建原核表达质粒pET-11c（＋）-stx2,经测序鉴定后转化E.col iBL21（DE3）,IPTG诱导表达,以SDS-PAGE、Western blot分析目的蛋白的表达.以EHEC O157：H7野生菌株作对照,通过对HeLa细胞的细胞毒作用及对小鼠攻毒实验,鉴定重组蛋白的生物学活性.结果 扩增的stx2基因约1230bp;原核表达质粒pET-11c（＋）-stx2经酶切和测序鉴定与预期序列一致;并在E.coli BL21（DE3）中得到表达,蛋白表达率约25%;PAGE初步测定目的蛋白的相对分子质量（Mr）约72×10^3;重组蛋白Stx2对HeLa细胞具有细胞毒作用,CD50为35 pg/ml;Stx2对小鼠致死剂量LD100为10μg.结论 成功克隆并表达了Stx2重组蛋白,为探讨O157：H7菌的感染机制及其防治研究奠定了一定的基础.
Objective To clone the gene and identify the activity of Shiga like toxin Ⅱ （ Stx2 ） of EHEC O157：H7. Methods The gene of stx2 was amplified from EHEC O157：H7 chromosome by PCR and then cloned into pMD18-T vector. Thereafter, the genes were cut from pMD18-T vector and cloned into prokaryotic expression plasmid pEr-lie（ ＋ ）, and the recombinant pasmids pET-11c（ ＋ ）-stx2 were transformed into E. coli BL21 （DE3）.The protein of Stx2 was analyzed and purified with polyacrylamide gel electrophoresis, Results The gene of six2 was successfully cloned into pET-11c（ ＋ ）. The molecular relative mass（M,） of the expressed products was 72× 10^3, the expression rate was about 25%, and the 50% cell death（ CD50 ）of Stx2 in HeLa cell was 35 pg/ml. Recombinant protein Stx2 showed lethal toxicity to mice when injected intraperitoneally, the LD100 was about 10 μg. Conclusion Stx2 was successfully expressed in prokaryotic expression system.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2006年第10期933-936,共4页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助(30500436)
军队杰出人才基金资助(04J009)
