德国小蠊Bla g5基因表达、纯化及生物信息学分析  

Expression,purification and bioinformatics analysis of the Bla g 5 gene of Blattella germanica

作　　者：季树宇 欧阳春艳 曹会 钟永浩 杨礼腾 刘晓宇[1] JI Shu-yu;OUYANG Chun-yan;CAO Hui;ZHONG Yong-hao;YANG Li-teng;LIU Xiao-yu(Health Science Center,Shenzhen University,Shenzhen 518060,China;Department of Allergy,the Third Affiliated Hospital of Shenzhen University,Shenzhen 518060,China)

机构地区：[1]深圳大学过敏反应和免疫学研究所,深圳518060 [2]深圳大学第三附属医院变态反应科,深圳518060

出　　处：《中国人兽共患病学报》2020年第12期975-981,987,共8页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.31729002、No.U1801286、No.81971514);广州市科学研究计划重点项目(No.201804020043);深圳市孔雀计划团队项目(No.KQTD20170331145453160);深圳市科技计划国际科技合作项目(No.GJHZ2018041819053);南山区“领航团队”支持计划项目(No.LHTD20180007);呼吸疾病国家重点实验室开放课题(No.SKLRD-OP-201909)。

摘　　要：目的德国小蠊(Blattella germanica)的分泌物、排泄物、蜕皮产物和尸体中携带含有大量过敏原,其中Bla g 5是引起过敏性疾病的一类重要过敏原。使用大肠杆菌克隆表达德国小蠊过敏原Bla g 5,然后使用过敏性疾病患者的血清IgE测试其变应原性。同时使用生物信息学预测Bla g 5基因的作用,了解到其分子特征。方法首先提取德国小蠊的总RNA,并根据已知的Bla g 5基因序列(GenBank:U92412)设计基因特异性引物。扩增其cDNA,并通过酶切连接构建表达载体pET-28a(+)-Bla g 5,然后转化入感受态细胞Rosetta(DE3)。选择单克隆阳性菌落进行扩增和培养。用0.1 mm/mol IPTG诱导目的蛋白的表达,然后通过金属螯合层析法纯化表达产物,并通过Western印迹法鉴定纯化产物的免疫学特性。结果对表达产物进行了十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),发现可溶性表达的Bla g 5蛋白的分子量约为25 kDa,与理论结果相符。纯化的重组蛋白的免疫印迹结果表明,Bla g 5可以特异性结合过敏患者的血清IgE。基于基因序列,我们预测了Bla g 5二级和三维结构、亲水性、可塑性、抗原性、表面可及性、同源性。发现其具有典型的谷胱甘肽硫转移酶(GST)特征。结论本研究中异源表达的德国小蠊Bla g 5过敏原蛋白对过敏患者的血清具有更高的结合活性,为相应的变应性疾病的诊断试剂和疫苗的开发研究奠定了基础。Blattella germanica contains many allergens in its secretions,excretions,molting products and corpses,among which Bla g 5 is an important allergen causing allergic diseases.Object:Here,we used E.coli to clone and express Blattella germanica allergen Bla g 5,and then used serum IgE from patients with allergic diseases to test its allergenicity.Bioinformatics was used to predict the role of the Bla g 5 gene and understand its molecular characteristics.Method:First,the total RNA of Blattella germanica was extracted,and gene-specific primers were designed according to the known Bla g 5 gene sequence(GenBank:U92412).The cDNA was amplified and ligated with a restriction endonuclease to construct the expression vector pET-28a(+)-Bla g 5,which was then transformed into Rosetta(DE3)competent cells.Monoclonal positive colonies were selected for amplification and culture.The expression of the target protein was induced with 0.1 mm/mol IPTG,the expressed product was purified with metal chelate chromatography,and the immunological properties of the purified product were identified by western blotting.Result:The expressed product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The molecular weight of soluble expressed Bla g 5 protein was approximately 25 kDa,in agreement with the theoretical results.The immunoblotting results of the purified recombinant protein showed that Bla g 5 specifically binds serum IgE in allergic patients.On the basis of the gene sequence,we predicted the secondary and three-dimensional structure,hydrophilicity,plasticity,antigenicity,surface accessibility and homology.The results indicated characteristics typical of glutathione S-transferase.Conclusion:The heterologous expression of German cockroach Blag5 allergen protein in this study indicated high binding activity to the serum of allergic patients,thus laying a foundation for the development of diagnostic reagents and vaccines for allergic diseases.

关 键 词：德国小蠊 过敏原 Bla g 5 免疫学鉴定 生物信息学分析 

分 类 号：R378.5[医药卫生—病原生物学]