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目的分别构建细粒棘球蚴(Eg)丝裂原活化蛋白激酶激酶MKK1和MKK2基因原核表达载体,诱导表达并纯化EgMKK1和EgMKK2蛋白。方法采集绵羊肝脏感染的Eg原头蚴,Trizol法提取原头蚴总RNA,反转录PCR分别扩增EgMKK1和EgMKK2基因片段,克隆至原核表达载体pET28a(+),经限制性内切酶双酶切和测序鉴定正确后转化大肠埃希菌感受态细胞BL21,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,采用镍柱亲和层析法纯化蛋白,并进行SDS-PAGE分析。结果测序证明pET28a-EgMKK1和pET28a-EgMKK2原核表达载体构建正确,在37℃经IPTG(终浓度1mmol/L)诱导可表达EgMKK1和EgMKK2蛋白,分子质量单位分别为44.96ku和64.01ku,与理论值相符,且以包涵体形式存在。表达产物纯化后的可溶性蛋白EgMKK1和EgMKK2含量分别为0.64mg/ml和1.2mg/ml。结论成功构建了MKK1和MKK2基因原核表达载体,表达蛋白可用于动物免疫,为研究EgMKK1和EgMKK2在Eg体内的生物学作用奠定了基础。
Objective To construct the prokaryotic expression vectors of mitochondria-derived activated protein kinase kinase (MKK1) and MKK2 (Echinococcus granulosus) respectively and induce the expression of EgMKK1 and EgMKK2 protein. Methods Eg prototheca infection in sheep liver and total RNA were extracted by Trizol method. The EgMKK1 and EgMKK2 gene fragments were amplified by reverse transcription PCR and cloned into prokaryotic expression vector pET28a (+). After restriction endonuclease di After digestion and sequencing, the recombinant plasmid was transformed into E.coli competent cells BL21 and induced by IPTG. The protein was purified by nickel column affinity chromatography and SDS -PAGE analysis. Results The prokaryotic expression vector pET28a-EgMKK1 and pET28a-EgMKK2 were constructed correctly. EgMKK1 and EgMKK2 were expressed by IPTG (final concentration 1mmol / L) at 37 ℃. The molecular mass units were 44.96ku and 64.01ku respectively, Match, and in the form of inclusion bodies. The content of purified protein EgMKK1 and EgMKK2 after purification was 0.64mg / ml and 1.2mg / ml, respectively. Conclusion The prokaryotic expression vectors of MKK1 and MKK2 genes were successfully constructed and the expressed proteins could be used for animal immunization. This study lays a foundation for studying the biological role of EgMKK1 and EgMKK2 in Eg.