中国农业科学2011，Vol.44Issue(17)：3641-3648,8.DOI:10.3864/j.issn.0578-1752.2011.17.017
福寿螺多功能纤维素酶基因egx的克隆及其体外功能性表达
Cloning and Functional Expression of a Multi-Functional Cellulase Gene egx from Mollusca, Ampullaria crossean in vitro
摘要
Abstract
[Objective] The objective of the study is to clone the multi-functional cellulase gene from Mollusca, Ampullaria crossean, and analyze its expression in vitro. [Method] The cDNA fragment was amplified by RT-PCR from the stomach of Mollusca, Ampullaria crossean, and then cloned into pMD18-T vector. Following sequencing, the gene was subcloned into the expression vector pET-32a (+) and pcDNA3.1 (+) using EcoR I and Not I restriction sites, and the enzymatic activity was determined by DNS. [Result] Sequence analysis showed that the 1 326 bp amplicon consists of 1 185 bp coding sequence and part of flanking sequence. The DNA sequence and the putative amino acid sequence shared 99% and 100% identity with the reported sequence, respectively. The purified product from E.coli BL-21 (DE3) showed hydrolytic activities to various substrates including carboxylmethyl cellulose sodium salt (CMC-Na), 2-hydroxyethyl-cellulose, hydroxyethyl-cellulose, sigmacell and xylan with specific activities of 24.78, 15.67, 18.42, 600.91 and 175.43 U·mg-1, respectiviely, while the recombination protein expressed in PK15 showed hydrolytic activities of 0.84, 0.78, 1.01, 14.62 and 4.23 U·mL-1, respectively. [Conclusion] The multi-functional cellulase from Mollusca, Ampullaria crossean, was cloned, functional expressed in pro- and eukaryotic cells, and this could provide a foundation for further research and application of the multi-functional cellulase gene from Mollusca, Ampullaria crossean.关键词
福寿螺/多功能纤维素酶/原核表达/真核表达/酶活测定Key words
Ampullaria crossean/ multi-functional cellulose/ prokaryotic expression/ eukaryotic expression/ emzyme assay引用本文复制引用
黄妙容,刘德武,吴珍芳..福寿螺多功能纤维素酶基因egx的克隆及其体外功能性表达[J].中国农业科学,2011,44(17):3641-3648,8.基金项目
国家科技重大专项(2008ZX08006004,2009ZX08006-012B) （2008ZX08006004,2009ZX08006-012B）
