摘要
目的:表达、纯化和鉴定法国梧桐花粉主要变应原基因Platanus acerifolia pollen allergen1(Pla a1)。方法:首先根据文献查找并在GenBank获取法国梧桐花粉主要变应原基因序列Pla a1,利用DNAStar软件进行密码子优化;合成全基因;将Pla a1与载体pET-44a连接后转入大肠杆菌Rosetta中进行诱导并优化目的蛋白表达;利用亲和层析法纯化该外源表达蛋白;应用Western blot,利用法桐花粉过敏患者血清鉴定纯化后的目的蛋白的抗原性。结果:成功构建了pET44a-Pla a1阳性质粒;获得了法桐花粉主要变应原重组蛋白Pla a1;对该重组蛋白进行了亲和层析纯化;免疫印记法表明重组蛋白具有一定的抗原性。结论:首次利用密码子优化的方法获得融合Strep TagⅡ的法桐花粉过敏原重组蛋白Pla a1,为制备高纯度变应原、重组低致敏过敏原及变应原核酸疫苗奠定基础。
Objective：To express and identify of the major allergen Pla a1 from Plane Tree Pollen. Methods：According to the literature of Plane Tree Pollen,the major allergen gene Pla a1 was obtained from the the GenBank;Codons optimized by DNA star software.The optimized gene was synthesized and cloned into the expression vector pET-44a,then transformed into E.coli Rosetta strain.The Pla a1 protein was induced by IPTG.Recombinant protein was purified by affinity chromatography and identified by Western blot. Results：The recombinant plasmid pET-44a-Pla a1 was successfully constructed.And then the recombinant Pla a1 protein was expressed and purified by affinity chromatography successfully.Western blot showed that the recombinant protein had some antigenicity. Conclusion： The recombinant Pla a1 protein was successfully expressed,purified and identified,which will provide basis for obtaining conveniently the highly purified allergens,recombinant hypo-allergenic allergens and allergenic DNA vaccines.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2012年第6期535-539,共5页
Chinese Journal of Immunology
基金
卫生部重点课题支气管哮喘重组变应原的特异性诊断和免疫治疗(No.2007353)
国家科技重大专项重大课题(2008ZX08011-005)及重点课题(2009ZX08011-004B)
国家自然科学基金项目(30771240)
广州市教育系统科研创新学术团队(B94118)资助
关键词
法国梧桐花粉
主要过敏原
密码子优化
表达
纯化
鉴定
Platanus Acerifolia Pollen
Major allergen
Codon optimization
Expression
Purification
Identification
