家蚕过敏原CSP5克隆表达、纯化鉴定及分析  

Immunological and bioinformatics analysis of a recombinant allergen of silkworm CSP5

作　　者：马一禾[1] 胡维[1] 梁志林[1] 王辉[1] 刘志刚[1] 

机构地区：[1]深圳大学过敏反应与免疫学研究所,深圳518060

出　　处：《现代免疫学》2016年第6期466-470,共5页Current Immunology

基　　金：国家自然基金(81273275);卫生部公益性行业科研专项(2015SQ00136);广东省对外科技合作项目(2013B051000088)

摘　　要：克隆家蚕过敏原CSP5（chemosensory protein 5 precursor）基因，表达、纯化该蛋白，鉴定其免疫活性，并进行生物信息学分析。人工合成家蚕化学感受蛋白5前体CSP5基因，将其连接至pMD18-T克隆载体，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达后进行纯化，用western blotting和ELISA鉴定其免疫学特性，并采用生物信息学方法预测CSP5蛋白的理化性质、三级结构、潜在B细胞抗原表位。本研究成功克隆了纯度较高的重组CSP5蛋白，测序鉴定蛋白分子质量约为14．25kD。重组过敏原CSP5能与家蚕过敏患者血清IgE发生特异性结合。生物信息学方法推导其潜在B细胞抗原表位为17～22、35～36、51～55、70～74、106～110、112～115。获得的重组蛋白CSP5具有与天然蛋白相似的免疫学活性，为以标准化抗原作为临床特异性诊断和治疗以及由家蚕引起的过敏性疾病的进一步研究奠定了基础。Cloning, expressing, purifying and testing the immunogenicity of chemosensory protein 5 precursor （CSPS）, and carrying out bioinformatics analysis of this protein. The gene coding for CSP5 was synthesized, and was then linked with the pMD18-T vector. The expression plasmid pET 32a-CSP5 was induced by IPTG. Purification of recombinant allergens of Bombyx Mori CSP5 proteins through the Ni ＋ affinity chromatography, western blotting and ELISA by immunological allergic patients＇ serum as the primary antibody. Predicting physical and chemical properties, three-tier structure, and potential B-cell epitope of CSP5 by bioinformatics methods. We obtained high purity recombinant CSP5 protein and identified by sequencing. The molecular mass of the expression product was about 14.5 kD. Western blotting results showed that recombinant allergen CSP5 could specifically bind with silkworm allergy serum IgE. Its potential B-cell epitopes were 17 N 22, 35-36, 51N 55,70 N 74,106N 110 and 112 - 115. The recombinant allergen CSP5 has the similar immunologic activity to natural CSP5 protein. These results lay the foundation for the specific diagnosis, treatment and further experimental studies of the silkworm allergy.

关 键 词：家蚕 CSP5蛋白 表达纯化 WESTERN BLOTTING 生物信息学分析 

分 类 号：R392.11[医药卫生—免疫学]