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目的长角血蜱(Hemaphysalis longicornis)不仅吸食家畜血甚至人血,还是多种疾病的传播媒介,对该蜱的研究具有重要的医学和兽医学意义。在大肠杆菌中表达长角血蜱的谷胱甘肽过氧化物酶(glutathione peroxidase)(HlGPx),为该酶在长角血蜱中的生理作用研究奠定基础,为该病媒的免疫防控提供参考。方法根据长角血蜱EST文库提示的片段序列的信息,设计引物采用PCR方法从长角血蜱成蜱的cDNA中扩增谷胱甘肽过氧化物酶基因。利用定点突变将该基因编码序列中一个编码硒半胱氨酸的密码子TGA突变为通用半胱氨酸密码子TGC,与表达载体pGEX-4T-1重组,转化BL21(DE3)pLysS,IPTG诱导表达,SDS-PAGE及免疫印迹分析。结果成功获得了长角血蜱谷胱甘肽过氧化物酶全长序列,突变后在大肠杆菌中诱导表达了约51kDa的重组蛋白,Western-blot显示表达产物与抗GST抗体有很强的交叉反应。结论突变的HlGPx可以在大肠杆菌中表达并可用于下一步制备免疫血清及功能分析。
Objective Hemaphysalis longicornis not only absorbs animal blood and even human blood, but also serves as a transmission medium for various diseases. The study of this tick has important medical and veterinary significance. Expression of glutathione peroxidase (HlGPx) in E. longicornis laid a foundation for the study of the physiological role of this enzyme in P. longicornis, for reference. Methods Based on the information of fragment sequence suggested by EST library of H. longicornis, primers were designed to amplify the glutathione peroxidase gene from the cDNA of ticks of ticks. A codon TGA encoding selenocysteine was mutated into a universal cysteine codon TGC by site-directed mutagenesis and recombined with the expression vector pGEX-4T-1 and transformed into BL21 (DE3) pLysS. IPTG induction Expression, SDS-PAGE and Western blot analysis. Results The full-length sequence of glutathione peroxidase was obtained successfully. After the mutation, a recombinant protein of about 51kDa was induced in E.coli. The result of Western-blot showed that the expressed product had strong cross-resistance with anti-GST antibody reaction. Conclusion The mutated HlGPx can be expressed in E. coli and can be used in the next step to prepare immune serum and functional analysis.