论文部分内容阅读
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 prepare a biologically active recombinant lethal factor 253 (LF253) antigen and to obtain a purified protein of interest for detection of its binding to lethal focfor (LF) protein against protective antigen , PA) capacity. Methods: The DNA of lethal factor LF253 fragment was amplified by PCR. The target gene was inserted into p ET-28a (+) expression vector. Using E. coli BL21 (DE3) as host strain, IPTG induced recombinant protein expression. The target protein was obtained by analysis. The protein antigenicity was determined by Western blot and ELISA. The binding affinity between the recombinant protein and the protective antigen PA was measured by Biacore T-100. The cytotoxicity was tested by biacore T-100. Results: The prokaryotic expression vector p ET-28a / LF253 was successfully constructed and the recombinant protein LF253 was induced. The results of Western blot and ELISA confirmed that the recombinant protein had good antigen specificity. The results of cytotoxicity showed that the recombinant protein could neutralize the biological effects of lethal toxin in vitro and in vivo. CONCLUSION: The fusion protein LF253 prepared in this study can bind with protective antigen PA, competitively inhibit the polymerization of LF full-protein and PA, and block the lethal effect of anthrax toxin, laying a foundation for the future development of anthrax vaccine and other drugs .