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目的克隆刚地弓形虫强毒RH株的表面抗原免疫作图蛋白1(Immune mapped protein-1,IMP1),并对其进行原核表达纯化、鉴定。方法采用逆转录法合成刚地弓形虫RH株的第一链c DNA,以此为模板,用PCR法扩增野生型IMP1基因的最大开放阅读框(ORF)序列,TA克隆测序鉴定后,插入原核表达载体p ET28b中,构建重组表达载体p ET28bIMP1,经双酶切和测序鉴定后转化大肠杆菌E.coli BL21(DE3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达携带6×组氨酸标签的IMP1融合蛋白,改变温度、诱导时间和IPTG浓度以优化诱导表达条件并测定蛋白可溶性,通过镍离子螯合(Ni2+-NTA)亲和层析纯化IMP1融合蛋白,经Western blotting验证重组蛋白的特异性。结果在弓形虫RH株速殖子c DNA中成功调取了IMP1基因的ORF序列,并通过TA克隆和测序鉴定了IMP1基因的正确性,在此基础上构建了原核表达重组质粒p ET28b-IMP1,经双酶切和测序验证了重组载体的正确性,并通过条件筛选确定了IMP1的最佳表达条件为20℃下0.3 mmol/L IPTG诱导9 h。经镍柱亲和层析纯化后,IMP1全蛋白表达量和可溶性均较好,与镍柱填料有较高的亲和力,能实现高效纯化。经SDS-PAGE及Western blotting鉴定,IMP1具有良好的免疫活性。结论新型强毒弓形虫RH株的表面抗原IMP1可在大肠杆菌原核表达系统中高效表达,全长蛋白可溶,性状稳定。本研究为进一步制备IMP1多克隆抗体,进行后续的体内表达研究以及抗弓形虫感染亚单位疫苗的构建和IMP1晶体结构研究奠定了基础。
Objective To clone Immune mapped protein-1 (IMP1) from Toxoplasma gondii RH strain RH strain and to purify and identify it by prokaryotic expression. Methods The first strand of c DNA of T. gondii RH strain was synthesized by reverse transcription. The ORF sequence of wild type IMP1 gene was amplified by PCR. After TA cloned and sequenced, The prokaryotic expression vector p ET28b was constructed and the recombinant expression vector p ET28bIMP1 was constructed. The recombinant plasmid p ET28bIMP1 was transformed into E.coli BL21 (DE3) by double enzyme digestion and sequencing. The recombinant plasmid pET28bIMP1 was transformed into E.coli BL21 (DE3) The IMP1 fusion protein carrying the 6 × histidine tag was induced to change the temperature, the induction time and the IPTG concentration to optimize the induced expression conditions and determine the protein solubility. The IMP1 fusion protein was purified by nickel ion chelation (Ni2 + -NTA) affinity chromatography The specificity of the recombinant protein was verified by Western blotting. Results The ORF sequence of IMP1 gene was successfully retrieved from tachyzoites RH strain tachyzoites and the correctness of IMP1 gene was identified by TA cloning and sequencing. Based on these, the recombinant plasmid p ET28b-IMP1 The correctness of the recombinant vector was confirmed by double enzyme digestion and sequencing. The optimal expression conditions of IMP1 were determined by conditional screening. The optimal conditions for the expression of IMP1 were induced by 0.3 mmol / L IPTG at 20 ℃ for 9 h. Purified by nickel affinity chromatography, IMP1 total protein expression and solubility are better, and nickel column packing has a higher affinity, to achieve efficient purification. The results of SDS-PAGE and Western blotting showed that IMP1 had good immunogenicity. Conclusion The surface antigen IMP1 of RH strain of virulent Toxoplasma gondii is highly expressed in E. coli prokaryotic expression system. The full-length protein is soluble and stable. This study laid the foundation for the further preparation of polyclonal antibody to IMP1, subsequent in vivo expression studies and construction of anti-Toxoplasma subunit vaccine and IMP1 crystal structure.