欧蓍草花粉主要过敏原Parj1的重组表达及鉴定

Cloning and expression of the Parietaria judaica pollen allergen Par j 1

目的:克隆、表达和纯化欧蓍草花粉主要过敏原Par j1。方法:根据Parj1.0102在GenBank中的序列号获得其核苷酸和氨基酸序列,确定开放阅读框,采用DNAstar软件优化密码子,合成全基因,并克隆到表达载体pET-44a中,转化表达宿主大肠杆菌Rosetta,优化蛋白表达条件并进行亲和层析纯化和Western blot鉴定。结果:PCR扩增及重组质粒测序结果表明成功地构建了pET-44a+/Par j1.0102原核表达质粒。对表达菌表达条件进行优化,最终确定在30℃,IPTG浓度为1.0 mmol/L,诱导4小时时蛋白表达量最高,重组蛋白经亲和层析纯化,SDS-PAGE分析纯化产物在23 kD处有明显的条带。Western blot表明重组蛋白具有与StrepII标签抗体结合活性。结论:国内首次获得融合StrepII标签的Par j1.0102重组蛋白,为欧蓍草花粉过敏诊断及特异性免疫治疗奠定基础。

Objective：To express and purify the Parietaria judaica pollen major allergen Par j 1.Methods：We obtained the nucleotide and amino acid sequence according to the published allergen number（O04404） in the GenBank.Codons optimized by DNAstar software.The optimized gene was synthesized and cloned into the expression vector pET-44a and transformed into E.coli Rosetta strain.Protein expression parameters were optimized on inducer concentration of IPTG,expression temperature and induction time.Recombinant protein was purified by affinity chromatography and identified by Western blot.Results：The recombinant plasmid pET-44a＋/Par j 1.0102 was successfully constructed which proved by PCR amplification and sequencing.Par j1.0102 protein was recombinantly expressed with StrepII tag under the best situation.Western blot showed recombinant protein could specifically bind to the StrepII tag antibody.Conclusion：The Par j 1.0102 protein is correctly expressed and the expressed protein could be used for diagnosis and immunotherapy of Parietaria judaica allergic patients with its allergenicity attenuated.