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目的利用大肠埃希菌(Escherichia coli)BL21(DE3)、Arctic Express(DE3)和Shuffle等3种不同的原核表达菌表达刚地弓形虫微线体蛋白2(Toxoplasma gondii microneme protein 2,TgMIC2),并进行蛋白质印迹(Western blotting)鉴定。方法参照Gen Bank中TgMIC2基因序列设计引物,以刚地弓形虫RH株速殖子RNA为模板,RT-PCR扩增获得DNA片段,PCR产物经NdeⅠ和HindⅢ双酶切后连至原核表达载体pET-30a(+),重组质粒转化入E.coli TOP10,双酶切筛选阳性质粒,将测序正确的阳性质粒pET30a-MIC2分别转化至E.coli BL21(DE3)、Arctic Express(DE3)和Shuffle表达菌,异丙基-β-D-硫代半乳糖苷诱导表达并优化各菌株的表达条件,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测表达产物。重组蛋白经镍柱纯化后,以抗组氨酸(His)单抗为一抗,Western blotting分析其免疫反应性。结果 TgMIC2基因的扩增产物长约2 000 bp。SDSPAGE结果显示,TgMIC2的相对分子质量(M_r)为80 000,在不同表达菌中的表达形式及表达量存在差异:TgMIC2在BL21(DE3)和Arctic Express(DE3)中均存在可溶性表达和包涵体表达,且在不同诱导条件下(15℃、16 h和37℃、4 h)可溶性蛋白均约占10%,表达量无明显差异;而TgMIC2在Shuffle中仅以包涵体形式表达。Western blotting分析结果显示,纯化后的可溶性TgMIC2和包涵体表达的TgMIC2重组蛋白都能被抗His的单抗识别。结论构建了pET30a-MIC2重组表达质粒,在3种不同的原核表达菌中成功表达TgMIC2重组蛋白。
Objective To express Toxoplasma gondii microneme protein 2 (TgMIC2) using three different prokaryotic expression strains Escherichia coli BL21 (DE3), Arctic Express (DE3) and Shuffle, And identified by Western blotting. Methods According to the sequence of TgMIC2 gene in GenBank, primers were designed and the tachyzoite RNA of Toxoplasma gondii RH strain was used as a template to amplify the DNA fragment by RT-PCR. The PCR product was double-digested with NdeI and HindIII and ligated into prokaryotic expression vector pET -30a (+). The recombinant plasmids were transformed into E.coli TOP10. The positive plasmids were screened by double enzyme digestion. The positive recombinant plasmids pET30a-MIC2 were transformed into E.coli BL21 (DE3), Arctic Express (DE3) and Shuffle The expression product of the strain was induced by isopropyl-β-D-thiogalactopyranoside and the expression conditions of the strains were optimized. The expression products were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Recombinant protein purified by nickel column with anti-histidine (His) monoclonal antibody as a primary antibody, Western blotting analysis of its immunoreactivity. Results The amplified product of TgMIC2 gene was about 2 000 bp in length. The results of SDSPAGE showed that the relative molecular mass (M_r) of TgMIC2 was 80 000, and there were differences in the expression and expression of TgMIC2 in different expression strains: TgMIC2 had soluble expression and inclusion bodies in BL21 (DE3) and Arctic Express (DE3) (15 ℃, 16 h and 37 ℃, 4 h), soluble protein accounted for about 10% of the total protein expression, while no significant difference was observed in TgMIC2 expression in Shuffle. Western blotting analysis showed that the purified soluble TgMIC2 and the recombinant TgMIC2 protein expressed in inclusion bodies could be recognized by anti-His monoclonal antibody. Conclusion The recombinant plasmid pET30a-MIC2 was constructed and the recombinant protein TgMIC2 was successfully expressed in three different prokaryotic expression strains.