法桐花粉主要过敏原基因Pla a1重组表达及鉴定

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。

Expression and identification of the major allergen gene Pla a1 from Platanus Acerifolia Pollen

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.