摘要
目的 :制备具有生物学活性的重组致死因子253(lethal factor 253,LF253)抗原,获得纯化的目的蛋白,检测其与全分子致死因子(lethal focfor,LF)蛋白竞争性结合保护性抗原(protective antigen,PA)的能力。方法:PCR扩增致死因子LF253片段的DNA,将目的基因插入p ET-28a(+)表达载体中,利用大肠杆菌BL21(DE3)作为宿主菌,IPTG诱导重组蛋白表达,通过His标签亲和层析柱获得目的蛋白,Western blot和ELISA法检测蛋白抗原性,Biacore T-100测定重组蛋白与保护性抗原PA结合的亲和力,细胞毒性实验检测其生物学活性。结果:成功构建原核表达载体p ET-28a/LF253,诱导获得重组蛋白s LF253的表达。Western blot和ELISA检测结果证实,该重组蛋白具有良好抗原特异性;细胞毒实验结果表明,重组蛋白可在体内外中和致死毒素引起的生物学效应。结论:本研究制备的融合蛋白s LF253能够与保护性抗原PA结合,可竞争性抑制LF全分子蛋白与PA的聚合,阻断炭疽毒素的致死作用,为今后炭疽疫苗等药物的研发奠定了基础。
Objective：To acquire the purified recombinant lethal factor 253（LF253）antigen which owned biological activity and test its competitive capacity binding protective antigen（PA）compared with lethal facfor（LF）protein. Methods：The LF253 gene was amplified by PCR,and the truncated LF gene was inserted into pET-28a （＋）,and transferred into E.coli.BL21 （DE3） as the host strain. LF253 was expressed as a recombinant protein induced with isopropyl---d-thiogalactoside （IVFG）. The protein was purified with His label affinity chromatography and was subjected to antigenicity by Western blot and ELISA. The affinity was detected by Biacore T-100,and the biological activity was detected by cellular toxicity test. Results：We successfully established prokaryotic expression vector pET-28a/LF253,and sLF253 was expressed and purified. Western blot and ELISA results showed that sLF253 had an excellent antigenicity. Cellular toxicity detection showed that recombinant protein neutralized the biological effects caused by lethal toxin in vitro and in vivo. Conclusion：Recombinant sLF253 can recognize PA and competitively inhibit LF polymerization with PA to block its lethal effects. This protein may lay the experimental basis for the future of anthrax vaccine research and development.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2015年第3期340-345,共6页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金项目(31170884)
江苏省自然科学基金项目(BK2012527)
关键词
炭疽
致死因子
表达
活性分析
anthrax
lethal factor
expression
activity analysis
