截短的弓形虫P30基因在E.coli中的高效表达及纯化条件的探索

HIGHLY EXPRESSION AND PURIFICATION FO TRUNCATED RECOMBINANT P30 OFTOXOPLASMA GONDII IN E.COLI

目的 构建能在E .coli中高效表达的弓形虫主要表面抗原 (P30 )基因的重组表达质粒 ,并对纯化条件进行优化。方法 对已知的弓形虫P30基因序列进行部分取舍 ,用PCR技术从弓形虫ZS1株的基因组DNA中扩增出截短的P30基因片段 ,插入载体pET 30 (a)中 ,转化大肠杆菌DH5α,IPTG诱导表达 ,包涵体经洗涤、变性、复性及不同程度的浓缩后 ,进行SDS PAGE及免疫印迹分析。结果 从弓形虫ZS1株基因组DNA中扩增出截短的P30基因片段 ,成功构建重组表达质粒 pET P30 ;SDS -PAGE显示蛋白表达带的分子量约为 31kD ,表达量占菌体总蛋白的 31.5 8% ,经 1M及 2M尿素洗涤后 ,其纯度分别达 6 3.42 %及 75 .74% ;免疫印迹显示 ,该纯化蛋白能被弓形虫病人阳性血清所识别 ,而且当浓缩至初始体积的 1/ 3～ 1/ 6时 ,纯化蛋白与DNA免疫鼠血清的反应最强。结论 成功构建重组质粒 pET P30 ,并以融合蛋白的形式进行了高效表达 ,经变性、复性后 ,该蛋白具有特异的免疫反应性 ,为弓形虫诊断试剂盒的研制打下基础。

Aim To construct recombinant plasmid containing the truncated major surface antigen P30 gene of T.gondii and express it in E.coli and optimized the purification condition.Methods The truncated P30 coding sequence was amplified from the genomic DNA of T.gondii ZS1 strain by PCR technique.Then,the amplified fragment was subcloned into the expression vector pET-30(a) and was expressed in E.coli.The recombinant protein was purified, refolded and condensed.Results The expression product was analyzed by SDS-PAGE. The result showed that the truncated P30 gene was highly expressed in E.coli,approximatedly 31.58% of whole bacterial lysate.The western blot analysis showed that the refolded recombinant P30 can be recognized by toxoplasma infective serum.The optimized condensation is 1/3 to 1/6 of original volume.Conclusion The truncated P30 gene of T.gondii has been expressed highly in E.coli as a fusion protein.After refolding,the recombinant P30 has a specific immunoactivity and could be used in immunological diagnosis.