粉尘螨铁蛋白的克隆、表达及蛋白特征分析
Cloning,expression and purification of dust mite ferritin and its molecular characteristics
摘要
Abstract
We obtained recombinant ferritin from Dermatophagoides f arinae ,and analyzed the characterization of the pro‐tein .A pair of primers was designed according to the known sequence of ferritin gene .The live mites identified and cultured lo‐cally were picked and the total RNA was extracted .The ferritin gene fragment was amplified by RT‐PCR ,and cloned into pET32a vector ,and then transferred into E .coli Top10 .The target gene obtained from the recombinant plasmid by digestion with Bam HⅠand Hind Ⅲ was connected to the prokaryotic expression vector pET‐32a .The expressed recombinant plasmid containing ferritin gene was constructed by cloning target gene into pET‐32a and transferred into E .coli Bl21 (DE3) .The ex‐pressed recombinant protein was analyzed by SDS‐PAGE ,and was purified by immobilized metal ion affinity chromatography (IMAC) .The ferritin expressed by dust mite was analyzed by the method of bioinformatics .The recombinant plasmid pET32a‐ferritin was constructed .SDS‐PAGE showed a correct molecular weight of the recombinant ferritin protein .After purification by affinity chromatography ,the protein showed only one strip on SDS‐PAGE gel .SDS‐PAGE showed a band at 20 kD .Dust mite ferritin contains 8 serine kinase ,7 threonine kinase ,7 tyrosine kinase ,and 0 histidine kinase phosphorylation sites .Hy‐drophilic region is larger than the hydrophobic region and it is an unstable protein .In conclusion ,the ferritin gene has been cloned and expressed .The purified ferritin has high purity . The study provides a basis for further study of composition and physicochemical properties of house dust mite allergen .关键词
粉尘螨/铁蛋白/蛋白表达/纯化/生物信息学分析Key words
Dermatophagoides f arinae/ferritin/ex-pression/purification/bioinformatics分类
医药卫生引用本文复制引用
林建立,詹政科,刘玉琳,范小琴,耿晓瑞,刘晓宇..粉尘螨铁蛋白的克隆、表达及蛋白特征分析[J].中国人兽共患病学报,2015,(10):927-930,937,5.基金项目
国家自然科学基金（No ．31328014、31400786）、广东省高等学校国际暨港澳台科技合作创新平台项目（No ．2012gjhz0009）、深圳市科技计划国际科技合作项目（GJHZ20130408174112021）、深圳市科技计划基础研究项目（No ．JCYJ20130329110735981、JCYJ20120613173233810）、深圳市南山区研发项目（No ．KC2012JSYB0003A）。Supported by the National Natural Science Foundation of China （Nos ．31328014 and 31400786）， the Guangdong  （）
Province Colleges and Universities International and Hong Kong ，Macao and Taiwan Science and Technology Cooperation Inno-vation Platform （No ．2012gjhz0009），the Shenzhen Science and Technology Project--International Scientific and Technological Cooperation Project （No ．GJHZ20130408174112021），the Basic Research Project of Shenzhen Science and Technology Project （Nos ．JCYJ20130329110735981 and JCYJ20120613173233810）， the Nanshan District Shenzhen Research and Development Project （）
