Abstract: Objective Objective To subclone，express and identify the immune mapped protein 1（IMP1）which encodes a surface an? tigen of Toxoplasma gondii. Methods Methods The cDNA of T. gondii RH strain was synthesized by reverse transcription PCR，the IMP1 open reading frame（ORF）was amplified by PCR using the T. gondii RH strain cDNA as template，the PCR products were identified by TA?cloning and sequencing，then the IMP1 ORF was subcloned into the NdeⅠand Xho I sites of the vector pET28b，and the positive recombinant pET28b?IMP1 was identified by double?digesting and sequencing. The protein of 6 × His tagged IMP1 was inducibly expressed in E. coli strain BL21（DE3）with isopropyl β?D?1?thiogalactopyranoside（IPTG），and the induction time，concentration of IPTG and temperature gradients to optimize protein expression conditions were determined. After the cells carried IMP1 were induced by the optimized conditions and harvested，the resulting bacteria were suspended in resuspension buffer and lysed by sonication，and the supernatants were loaded onto the Ni 2+ Chelating Sepharose Fast Flow col? umn for affinity chromatography of the N?terminal 6 × His tagged IMP1 protein. Finally，the fusion IMP1 proteins were identified by Western blotting. Results Results The ORF sequence of IMP1 was successfully subcloned from the cDNA of Toxoplasma Gondii RH strain，and the amplified product was sequenced and identified，based on which the IMP1 ORF gene was inserted into the prokaryotic expression vector pET28b，and the recombinant pET28b?IMP1 was constructed successfully. The double?digesting and sequencing results indicated the validity of the recombinant vector. And the optimized conditions for the expression of IMP1 was determined，namely 0.3 mmol/L IPTG induction for 9 h at 20 ℃. Furthermore，IMP1 protein was expressed solubly and che? lated on Ni 2 + sepharose beads with high affinity，thus this protein could be purified efficiently by affinity chromatography. The pure fusion protein was confirmed with fine immunocompetence by SDS?PAGE and Western blotting. Conclusions Conclusions IMP1 protein can be high efficiently expressed by the E. coli prokaryotic expression systems，the protein of IMP1 is soluble and has stable characters. The study may lay a useful foundation for the following works including in vivo expression of IMP1，crystal structure study of IMP1 and anti?toxoplasmosis subunit vaccine development.

Key words: Toxoplasma gondii； IMP1 gene； Surface antigen； Prokaryotic expression； Purification

摘要： 目的 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 方法 采用逆转录法合成刚地弓形虫RH株的第一链cDNA， 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pET28b中， 构建重组表达载体pET28b? IMP1， 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 （Ni 2+ ?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 结果 在弓形虫RH株速殖子cD? NA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pET28b?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 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。

关键词: 弓形虫； 免疫作图蛋白1基因； 表面抗原； 原核表达； 纯化