CAJ | 학술논문

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（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。
목적：극륭강지궁형충강독RH주적표면항원면역작도단백1（Immune mapped protein?1，IMP1），병대기진행원핵표체순화、감정。방법채용역전록법합성강지궁형충RH주적제일련cDNA，이차위모판，용PCR법확증야생형IMP1기인적최대개방열독광（ORF）서렬，TA극륭측서감정후，삽입원핵표체재체pET28b중，구건중조표체재체pET28b?IMP1，경쌍매절화측서감정후전화대장간균E.coli BL21（DE3），경이병기류대?β?D?반유당감（IPTG）유도표체휴대6×조안산표첨적IMP1융합단백，개변온도、유도시간화IPTG농도이우화유도표체조건병측정단백가용성，통과얼리자오합（Ni2+?NTA）친화층석순화IMP1융합단백，경Western blotting험증중조단백적특이성。결과재궁형충RH주속식자cD?NA중성공조취료IMP1기인적ORF서렬，병통과TA극륭화측서감정료IMP1기인적정학성，재차기출상구건료원핵표체중조질립pET28b?IMP1，경쌍매절화측서험증료중조재체적정학성，병통과조건사선학정료IMP1적최가표체조건위20℃하0.3 mmol/L IPTG유도9 h。경얼주친화층석순화후，IMP1전단백표체량화가용성균교호，여얼주전료유교고적친화력，능실현고효순화。경SDS?PAGE급Western blotting감정，IMP1구유량호적면역활성。결론신형강독궁형충RH주적표면항원IMP1가재대장간균원핵표체계통중고효표체，전장단백가용，성상은정。본연구위진일보제비IMP1다극륭항체，진행후속적체내표체연구이급항궁형충감염아단위역묘적구건화IMP1정체결구연구전정료기출。
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 pro? tein 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.