Cloning,expression and purification of dust mite ferritin and its molecular characteristics
10.3969/j.issn.1002-2694.2015.10.007
- VernacularTitle:粉尘螨铁蛋白的克隆、表达及蛋白特征分析
- Author:
Jianli LIN
;
Zhengke ZHAN
;
Yulin LIU
;
Xiaoqin FAN
;
Xiaorui GENG
;
Xiaoyu LIU
- Publication Type:Journal Article
- Keywords:
Dermatophagoides f arinae;
ferritin;
ex-pression;
purification;
bioinformatics
- From:
Chinese Journal of Zoonoses
2015;(10):927-930,937
- CountryChina
- Language:Chinese
-
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 .
