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

发布时间：2018-04-29 12:09

本文选题：粉尘螨 + .kDa溶菌酶(Bac)　；参考：《中国人兽共患病学报》2016年09期

【摘要】：目的克隆表达粉尘螨13.8kDa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396bp,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14kDa。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。
[Abstract]:Objective to clone and express the 13.8kDa lysozyme gene of Dermatophagoides farinae, purify and identify the allergen of the protein and analyze it by bioinformatics. Methods Total RNAs were extracted from pure cultured Dermatophagoides farinae and primers were designed according to known gene sequences. Bac gene fragments were amplified by RT-PCR and the products were linked into pMD-32T vector. After amplification, the target gene fragment was ligated to the expression vector of Pet-44a by restriction endonuclease EcoRI and XhoI digestion, and transformed into E. coli BL21DDE3) for expression induced by IPTG. Indirect ELISA assay to detect the specific allergen of recombinant Bac protein clustalw2 homology analysis was carried out to construct a phylogenetic tree named ProtParam Tools to predict its physical and chemical properties. PSIPRED predicted its secondary structure and SWISS-MODEL predicted its tertiary structure. The T cell epitopes of Bac protein were predicted by using the relative software of IEDB and preprod. B cell epitopes of Bac were predicted by DNAStar. Results the Bac gene of Dermatophagoides farinae was cloned successfully by sequencing, and its open reading frame was 396bp, encoding 131amino acids. The Bac gene was introduced into E. coli E. coli BL21 (DE3), and the recombinant Bac protein was highly expressed by IPTG induction. The protein mainly exists in soluble form and its molecular weight is about 14 kDa. Indirect ELISA assay demonstrated that Bac could bind to serum IgE in patients with Dermatophagoides farinae allergy. Sequencing showed that the cloned Bac gene of Dermatophagoides farinae in our laboratory had 96 homology with GenBank KF113885.1 published on GenBank. Phylogenetic tree results showed that the genetic relationship between Dermatophagoides farinae and housedust mites was close. The prediction of physicochemical properties showed that Bac protein was stable. The prediction results of secondary and tertiary structures showed that the structure of Bac was mainly predicted by irregular curl composition. T cell epitopes. Four peptide sequences 6-1438-46-45-99122-130. B cell epitopes were predicted and 6 peptide sequences were predicted, namely 15-30CG-4044-54-58-735-110101-116N. Conclusion Bacillus farinae was cloned and expressed successfully, and the recombinant Bac protein had good immunogenicity, which laid a theoretical foundation for further study on the structure and physical and chemical properties of dust mite allergen.
【作者单位】：深圳大学过敏反应和免疫学研究所;
【基金】：国家自然科学基金(No.91442118、No.31400786) 广东省科技计划社会发展项目(No.2013B03180002) 广东省对外科技合作项目(No.2013B051000088) 广东省公益研究与能力建设专项资金(No.2013B031800023) 深圳市科技计划国际科技合作项目(No.GJHZ20130408174112021) 深圳市科技计划基础研究项目(No.JCYJ20140828163633992)联合资助~~
【分类号】：R384.4

【相似文献】