反枝苋花粉泛过敏原profilin基因的克隆表达、纯化及免疫学鉴定  

Cloning,Expression and Immune Characterization of Panallergen Profilin from Amaranthus Retroflexus Pollen

作　　者：谢水祥[1] 龚祥 龚苗[3] 马莉[3] 肖小军[3] 刘志刚[3] 

机构地区：[1]赣南医学院病原生物学教研室,江西赣州341000 [2]南昌大学科学技术学院,2011级南昌330029 [3]深圳大学过敏反应与免疫学研究所,广东深圳518060

出　　处：《南昌大学学报（医学版）》2014年第12期4-8,共5页Journal of Nanchang University：Medical Sciences

基　　金：国家自然科学基金(811160129;81302553);广东省工程技术研究开发中心项目(2013158925);广东省高等学校国际暨港澳台科技合作创新平台项目(2012gjhz0009)

摘　　要：目的克隆表达反枝苋花粉中泛变应原肌动蛋白抑制蛋白(profilin),并鉴定其免疫学活性。方法利用RT-PCR结合RACE技术克隆反枝苋花粉泛变应原profilin的全长基因,并进行序列分析。设计带有酶切位点的特异性引物,采用RT-PCR获得整个反枝苋花粉profilin的开放阅读框,将其与p ET28a载体连接并转化大肠杆菌BL21(DE3)进行诱导表达,Ni2+亲和层析柱纯化重组蛋白,采用Western-blot检测其Ig E结合活性。结果 c DNA核苷酸测序表明反枝苋花粉profilin的全长基因由675个碱基组成,开放阅读框为399 bp,编码131个氨基酸。重组反枝苋花粉profilin在大肠杆菌中可溶性表达,进一步经Western-blot检测具有良好的Ig E结合活性。结论成功地克隆和表达了反枝苋花粉profilin,并具有良好的Ig E应答免疫活性。Objective To clone and express Amaranthus retroflexus pollen profilin,and to evaluate the immunologic activity of the allergen. Methods The full-length profilin gene from Amaranthus retroflexus pollen was cloned using RT-PCR and RACE. Then the sequence was analyzed. The open reading frame（ ORF） of panallergen profilin was amplified by RT-PCR using specific primers with restriction sites. The profilin gene was cloned into p ET28 a vector,expressed in E. coli BL21（ DE3）,and purified by Ni2 ＋affinity chromatography. The Ig E-binding capacity of the recombinant protein was analyzed by Western-blot. Results Nucleotide sequencing of the c DNA revealed that the cloned fragment of Amaranthus retroflexus pollen profilin was 675 bp in length with a 399 bp ORF encoding 131 amino acid residues. The recombinant protein was expressed in E. coli as a soluble protein which showed a strong IgE reactivity.Conclusion The profilin from the Amaranthus retroflexus pollen was successfully cloned and expressed with a strong IgE reactivity.

关 键 词：反枝苋花粉 抑制蛋白 基因表达 纯化 

分 类 号：R392.8[医药卫生—免疫学]