葎草花粉主要变应原Hum s1的表达、纯化与结构预测  

Expression,Purification and Structure Prediction of Hum s1,a Major Allergen of Humulus Scandens

作　　者：刘昀[1] 孙秀珍[1] 徐晶[1] 吴媛媛[1] 

机构地区：[1]西安交通大学第二附属医院呼吸科,西安710004

出　　处：《四川大学学报（医学版）》2016年第1期14-18,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(No.81102252)资助

摘　　要：目的克隆、表达、纯化葎草花粉主要变应原Hum s1,预测、分析其蛋白结构及功能,为评估该重组变应原的诊断及治疗效果奠定基础。方法采用PCR扩增目的基因Hum s1,并将Hum s1通过KpnⅠ/XhoⅠ位点克隆到pET32a表达载体,转化到克隆菌株E.coli DH5α,用PCR验证重组载体,并将表达载体转化至表达菌株E.coli BL-21,IPTG诱导表达目标基因,利用快速蛋白液相色谱技术纯化融合蛋白,并对表达蛋白的结构与功能进行预测分析。结果成功构建原核表达质粒pET32a-G2,并表达纯化葎草花粉主要变应原Hum s1,纯度达90%以上。分析其结构含有3个潜在的抗原表位,2个EF-手型结构域,成功构建其三维模型。ELESA试验证明重组蛋白能与葎草花粉过敏患者血清结合,具有免疫学活性。结论成功构建了葎草花粉变应原Hum s1的原核表达载体,表达并纯化了重组蛋白,并通过生物信息学方法获得了目的蛋白的抗原表位和三维结构模型,为进一步开展重组疫苗研究奠定了基础。Objective Hum s1,a major allergen of Humulus Scandens,was cloned,expressed and purified.Its protein structure and function was predicted and analyzed,which provided a foundation for further studies into the diagnostic and therapeutic efficacy of the recombinant allergen.Methods The target gene was amplified by PCR and sub-cloned into the expression vector pET32 athrough KpnⅠ/XhoⅠ site.The recombinant plasmid was transformed into clone strain E.coli DH5α.The positive recombinant plasmid identified by PCR was transformed into the expression strain E.coli BL-21.The expressed fusion protein was induced by IPTG,and purified using fast protein liquid chromatography.The structure and function of the protein was predicted and analyzed.ResultsProkaryotic expression plasmid pET32a-G2 was constructed successfully.Hum s1 was expressed and purified.The purity of expressed fusion protein exceeded 90%.It has three potential antigen epitopes and two EF-hand structural domains.A three-dimensional model was successfully constructed.The recombinant protein was proved to have immunological activity,with ELESA showing good attachment to sera samples of patients who were allergic to Humulus Scandens.Conclusion Prokaryotic expression vector of Hum s1 was successfully constructed.The recombinant protein was expressed and purified,with its epitope and three-dimensional model being predicted by bioinformatics.The study provided a basis for further development of recombinant vaccines.

关 键 词：葎草花粉 重组变应原 基因克隆 

分 类 号：R562.25[医药卫生—呼吸系统] Q943.2[医药卫生—内科学]