弓形虫表面抗原IMP1的克隆原核表达及免疫学鉴定  

Cloning,prokaryotic expression and immunological identification of Toxoplasma surface antigen IMP1

作　　者：寇景轩[1] 赵桂华[1] 魏庆宽[1] 徐超[1] 朱嵩[1] 尹昆[1] 

机构地区：[1]山东省医学科学院,山东省寄生虫病防治研究所,济宁272033

出　　处：《中国血吸虫病防治杂志》2015年第3期285-289,共5页Chinese Journal of Schistosomiasis Control

基　　金：国家自然科学基金(31300617);山东省优秀中青年科学家科研奖励基金(BS2013SW015);山东省医药卫生科技发展计划项目(WSO370)

摘　　要：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 subclone express and identify the immune mapped protein 1 IMP1 which encodes a surface an-tigen of Toxoplasma gondii. 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 isopropylβ-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 Ni2＋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 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 IMP 1 was determined namely 0.3 mmol/L IPTG induction for 9 h at 20℃. Furthermore IMP1 protein was expressed solubly and che-lated on Ni2＋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 IMP1

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 

分 类 号：R382.5[医药卫生—医学寄生虫学]