重组扁豆过敏原Lenc 1表达纯化及免疫活性鉴定

Purified Expression and Identification of Immune Activity of the Major Allergen Len c 1 of Lentil

目的:纯化重组扁豆过敏原Len c 1(rLen c 1)并进行免疫活性鉴定。方法:通过原核表达的方式生产rLen c 1,然后采用亲和层析的方法纯化带有Strep II标签的目的蛋白。将BALB/c小鼠随机分为对照组(注射生理盐水)和过敏原致敏组(注射rLen c 1),通过腹腔注射方式免疫小鼠,建立BALB/c小鼠扁豆致敏模型,利用间接ELISA法检测血清总IgE和过敏原特异性IgE,鉴定重组扁豆过敏原的免疫活性。结果:IPTG成功诱导Len c 1蛋白表达,rLen c 1蛋白主要以包涵体形式表达,通过透析复性和亲和层析获得纯化的复性扁豆过敏原蛋白rLen c 1,成功建立小鼠致敏模型。和对照组相比,致敏组小鼠TIgE及过敏原特异性IgE水平均明显增高,结论:获得纯化的具有免疫活性的rLen c 1过敏原蛋白,为扁豆过敏机制研究、单克隆抗体的制备、以及临床诊断和免疫治疗奠定基础。

Objective： To purify recombinant lentils allergens Lenc 1 （rLen c 1） and identify the immune activity. Methods： By means ofprokaryotic expression system rLen c 1 was produced, then the target protein with Strep II tag was purified with the method of affinity chromatography. BALB/e mice were randomly divided into control group （injected by normal saline） and allergen sensitized group （injected by rLen c 1）. The mice were immuned by intmperitoneal injection to set up BALB/c mice lentils allergic model, then the total IgE and allergen specific IgE were detected by indirect ELISA to identify the immune activity of recombinant lentil allergen. Results： Lenc 1 protein expression was successfully induced by IPTG, which was expressed in the form of inclusion body; with dialysis renaturation and affinity chromatography,the highly purified renatured protein rLen c 1 was gained, and mice sensitization model was established successfully. Compared with the control group of mice, the TIgE and allergen specific IgE significantly increased in sensitized group of mice. Conclusions： Purified rLen c 1 allergen protein with immune activity was obtained to lay a foundation for the preparation ofmonoclonal antibodies, clinical diagnosis and immunotherapy.