害嗜鳞螨过敏原Lep d2的原核表达与纯化  

Expression and purification of the major allergen Lep d 2 of Lepidoglyphus destructor

作　　者：关志澳 何颖[1] 邹泽红[1] 陶爱林[1] 

机构地区：[1]广州医科大学附属第二医院、广东省过敏反应与免疫重点实验室/变态反应国家临床重点专科/呼吸疾病国家重点实验室变态反应研究室,广东广州510260

出　　处：《热带医学杂志》2016年第6期691-694,698,共5页Journal of Tropical Medicine

基　　金：国家科技重大专项重点课题(2014ZX0801105B);国家临床重点专科建设项目(520102-110);广东省产学研项目(2013B090500129)

摘　　要：目的构建害嗜鳞螨(Lepidoglyphus destructor)过敏原Lep d 2的原核表达系统,获得Lep d 2的重组纯化蛋白。方法通过双酶切pUC-Lep 2质粒,获取基因Lep d 2,并结合高保真PCR获取去信号肽基因rLep d 2,连接载体pET44a并转入表达菌株Rosetta;利用异丙基-β-D-硫代半乳糖苷(IPTG)和蛋白免疫印迹法分别进行诱导表达与鉴定;用盐酸胍溶解包涵体并进行透析复性;使用Strep II亲和填料来纯化目的蛋白。结果构建了Rosetta-rLep d 2的原核表达体系;rLep d 2蛋白在菌体内主要表达形式为包涵体,通过透析复性和亲和纯化获得了高纯度的rLep d 2蛋白。结论获得的重组rLep d 2蛋白可为下一步评价其过敏原性提供研究材料。Objective To obtain purified recombinant allergen Lep d 2 of Lepidoglyphus destructor by prokaryotic expression system. Methods The target gene of Lep d 2, and rLep d 2 the gene without signal peptide were obtained with double enzyme digestion and high fidelity PCR of pUC-Lep 2 plasmid, and then were ligated to the p ET44a vector and transformed into Rosetta. The protein was expressed by IPTG induction and identified by Western blot. The inclusion body of rLep d 2 was denatured in guanidine hydrochloride and refolded by gradient dialysis, and finally the protein was purified by Strep II tag affinity chromatography. Results The expression system of Rosetta-rLep d 2 was constructed.The rLep d 2 protein was expressed as inclusion body, and the highly purified protein rLep d 2 was obtained by dialysis renaturation and affinity chromatography. Conclusion The recombinant protein rLep d 2 can be used to study its allergenicity for the future.

关 键 词：LEP d 2 害嗜鳞螨 原核表达 蛋白纯化 

分 类 号：R392.11[医药卫生—免疫学]