摘要
目的克隆表达肠出血性大肠杆菌(EHEC)O157∶H7志贺样毒素B亚单位(Shiga liketoxin2B,Stx2B),并对其初步纯化。方法设计引物采用PCR法自EHEC O157∶H7基因组扩增志贺样毒素B亚单位的编码基因stx2b,T-A克隆后构建原核表达质粒pET-22b(+)-stx2b,经测序鉴定后转化E.coliBL21(DE3),IPTG诱导表达,以Tricine-PAGE和Western blot分析目的蛋白的表达,并采用固相镍离子亲和层析纯化重组蛋白。结果扩增的stx2b基因约210bp;原核表达质粒pET-22b(+)-stx2b经酶切和测序鉴定与预期序列一致;并在E.coliBL21(DE3)中得到高效可溶性表达,蛋白表达率约30%;PAGE初步测定目的蛋白的相对分子质量(Mr)约7×103;经固相镍离子亲和层析纯化后,蛋白纯度可达90.1%。结论EHEC O157∶H7志贺样毒素B亚单位的高效表达与初步纯化,有利于进一步的生物学性质研究。
Objective To clone the germ of Shiga like toxin 2B of EHEC O157 ： H7. Methods The stx2b germ was amplified from EHEC O157：147 chromosome by PCR and then cloned into pMD18-T vector. Thereafter, the gene was cut from pMD18-T vector and cloned into prokaryotic expression plasmid pET-22b（ ＋ ）. Then the recombinant pasmids were transformed into E. coli BL21（DE3）. The Stx2B protein was analyzed and purified with polyacrylamide gel electrophoresis and nickel-affinity chromatography coloum. Results The stx2b gene was successfully cloned into pET-22b（ ＋ ）. The molecular mass（ Mr ） of the expressed product was 7× 10^3 ; the expression rate was about 30%, and the purity was 90.1%. Conclusion The stx2b has been 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2期155-158,共4页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目(30500436)
军队杰出人才基金资助项目(04J009)
