重组泡球蚴Em18蛋白不同纯化方法的比较

目的：比较不同方法纯化重组泡球蚴Em18的效果。方法：进一步对BL21-pET41a-Em18重组菌的诱导表达条件进行摸索优化,采用改良的超声程序破碎大肠杆菌细胞,分别经不同的纯化方法进行蛋白纯化,通过SDS-PAGE对纯化结果进行比较分析,Western blot进行活性鉴定。结果：①在菌液OD600为0.8-1.0,加IPTG终浓度为1 mmol/L,37℃诱导3h,rEm18-GST重组蛋白得到成功高表达,在相对分子量为50kDa处有表达条带。②超声程序为：工作3 s,间歇4s,功率200~300W,超声体系中加入溶菌酶和蛋白酶抑制剂作用,可促进大肠杆菌细胞的破碎,降低目的蛋白的降解;加入终浓度为1%的Triton-X100作用可增加融合蛋白的可溶性。③通过比较不同方法纯化重组泡球蚴Em18的效果表明采用单纯His柱纯化可获得浓度高、纯度高的rEm18-GST重组蛋白。Western blot分析表明该蛋白能与泡型包虫病（AE）患者血清特异性反应。结论：建立了一种纯化原核表达Em18融合蛋白的较为经济和有效的方法,得到大量有生物学活性的Em18融合蛋白,为包虫病诊断试剂盒的研制奠定基础。

Comparison of Different Methods to Purify Recombinant Em18 Antigen for Immunodiagnosis of Echicoccosis

Objective: To compare the purification effects of rEm18 by different methods. Methods: pET41a-Em18 in E. coli BL21 were expressed when induced by IPTG, E, coli cells were broken by different procedure of sonication, further purified respectively by different purified methods. The purified rEm18 was analyzed by SDS-PAGE and Western blot. Result: ①The pET41a-Em18 recombinant protein induced with 1.0 mmol/L IPTG at 37℃ for 3 h can be detected as a band of 50kDa by SDS-PAGE. ②The schizolyzed promotion and degradation reduction of E. coli cell was optimized by adding lysozyme and proteinase inhibitor during sonication process and the solubility of rEm18 fusion protein was increased by using Triton-X100. The optimized procedure of sonication: work 3s, rest 4s.③Compared three different purified methods and optimized concentration, pH in the different buffer, higher efficiency and more purified protein was obtained by the His resin and western blot analysis indicated that rEm18 fusion protein could react specifically against a pool of sera from AE patients. Conclusion: A more efficiency and economic purified method of the Em18 fusion protein was established to develop an immunodiagnosis kit of Echicoccosis.