粉尘螨13.8 kDa溶菌酶的克隆表达、纯化鉴定及生物信息学分析

目的 克隆表达粉尘螨13.8 kDa 溶菌酶(Bac)基因, 纯化鉴定蛋白的过敏原性, 并进行生物信息学分析。方法 挑取经纯培养的粉尘螨,提取总RNA,根据已知基因序列设计引物,经RT-PCR扩增Bac基因片段,产物连入pMD-32T载体中。扩增后,利用限制性内切酶EcoRI和XhoI双酶切将目的基因片段连接到Pet-44a表达载体上,转化到大肠杆菌BL21( DE3)中经IPTG诱导表达;间接ELISA法检验重组Bac蛋白特异性过敏原性;clustalw2进行同源性分析,MEGA5工具包来构建系统进化树,ProtParam Tools 预测其理化性质,PSIPRED预测其二级结构,SWISS-MODEL预测其三级结构。利用网络服务器 IEDB内相关软件和Preprod,对Bac蛋白T细胞抗原表位进行预测。用DNAStar对Bac的B细胞抗原表位进行预测。结果 经测序鉴定,本研究成功克隆出了粉尘螨Bac基因,其开放阅读框396 bp,编码131组氨基酸。将Bac基因导入大肠杆菌E.coliBL21 (DE3),经IPTG诱导后高效表达Bac重组蛋白。该蛋白主要以可溶性形式存在,蛋白分子量约14 kDa。间接ELISA法证明Bac能与粉尘螨过敏患者血清IgE结合。测序发现本实验室克隆的粉尘螨Bac基因与GenBank上公布的GenBank KF113885.1同源性为96%。系统进化树结果显示粉尘螨与屋尘螨亲缘关系比较近。理化性质预测显示Bac蛋白质较稳定。二级结构及三级结构预测结果显示Bac的结构主要以无规则卷曲组成。T细胞抗原表位预测得到 4个肽序列(6-14、38-46、85-99、122-130)。B细胞抗原表位预测得到6个肽序列(15-30、26-40、44-59、58-73、95-110、101-116)。结论 成功克隆并表达了粉尘螨Bac,并证实重组Bac蛋白具有良好的免疫原性,为进一步研究尘螨过敏原的结构成分及其理化性质奠定理论基础。

Cloning and expressing the Dermatophagoides farinae 13.8 kDa lysozyme (Bac) gene,purifying and identifying the protein allergenicity and analysis of the Bac protein

The aim for this study is cloning and expressing the Dermatophagoides farinae 13.8 kDa lysozyme (Bac) gene, purifying and identifying the protein allergenicity and conducting bioinformatics analysis of the Bac protein. Total RNA was extracted from cultured dust mites. The primers designed according to sequences was amplified by RT-PCR. The PCR product was cloned into pMD32-T vector, and then subcloned into pET-44a vector by restriction endonuclease EcoR I and Xho I. The recombinant plasmid pET44a-Bac was transformed into E. coli BL21 and was induced with IPTG. Enzyme-linked immunosorbent assay (ELISA) technique was used to test the allergen of Bac and the clustalw2 was used to conducted homology analysis and MEGA5 to construct phylogenetic tree of Bac. ProtParam tools were used to predict its physical and chemical properties. PSIPRED and SWISS-MODEL were used to predict its secondary structure and tertiary structure. IEDB and Preprod was used to analyze the T cell antigen epitope. DNAStar was used to analyze the B cell antigen epitope of Bac. After sequencing, we have successfully cloned the Bac gene, and the open reading frame was 396 bp, encoding 131 amino acid group.The Bac gene was transferred into E. coli BL21 (DE3). After induction with IPTG, Bac protein was largely expressed. The protein mainly existed in soluble form; protein molecular weight was about 14 kDa. The indirect ELISA method results showed that Bac could bind with dust mite allergic patients serum IgE. Comparing the cloned Der f Bac amino acid sequence with other mites, we found that its homology with NCBI accession number KF113885.1 was 96%. The molecular evolutionary tree analysis showed that Bac had a close relationship with Dermatophagoides pteronyssinus. The physical and chemical properties prediction showed Bac protein was stable. The prediction of the secondary and tertiary structure indicated that Bac mainly consisted of random coil. Bioinformatics analysis predicted four peptides (6-14, 38-46, 85-99, 122-130) as the T cell epitopes and six peptides (15-30, 26-40, 44-59, 58-73, 95-110, 101-116) as the B cell epitopes. We have successfully cloned and expressed the Bac, and confirmed that the recombinant Bac protein has good immunogenicity. The study provides a basis for further study of composition and physicochemical properties of house dust mite allergen.