家蝇几丁质结合蛋白(Md-CBPⅠ)的表达纯化及活性分析

Expression，purification and activity analysis of a chitin binding protein (Md- CBPⅠ)from musca domestica

关键词:  家蝇,几丁质结合蛋白,克隆,原核表达

Key words:  Musca domestica  Chitin binding protein  Clone  Prokaryotic expression,

摘要:

本研究采用PCR技术对鸡源沙门氏菌诱导家蝇幼虫构建的抑制性消减文库(SSH)中筛选到的的家蝇幼虫几丁质结合蛋白Ⅰ基因(Musca domestica chitin binding proteinⅠ,Md-CBPⅠ)进行扩增,并成功构建了重组表达质粒pET-32a-Md-CBPⅠ,于大肠杆菌BL21(DE3)中得以高效表达,纯化并获得了Md-CBPⅠ融合蛋白。进一步对该蛋白的亲和活性进行了研究,现Md-CBPⅠ融合蛋白对几丁质以及纤维素均有一定的结合作用,且其对几丁质的结合作用相对较好,上述研究为家蝇几丁质结合蛋白生物学活性和免疫学活性的研究奠定了基础。

Abstract:

Musca domestica chitin binding proteinⅠ(Md-CBPⅠ)was screened from suppression subtractive library(SSH) in Musca domestica larvae induced by Salmonella pullorum of chickens and also amplified by PCR techniques. The recombinant expression plasmid of pET-32a-Md-CBPⅠ was constructed successfully and expressed in E.Coli BL21(DE3). fusion protein was obtained by purification,further more, the preliminary study was made on the affinity of which revealed that the fusion protein has binding affinity of both chitin and cellulose, and the affinity of chitin was stronger than cellulose. This research laid a foundation for further research on biological activity and immunological activity of Md-CBPⅠ.