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

doi:10.3864/j.issn.0578-1752.2011.17.017

摘要/Abstract

摘要： 【目的】克隆福寿螺多功能纤维素酶基因egx，并在大肠杆菌和哺乳动物细胞中进行表达分析。【方法】通过RT-PCR的方法，从福寿螺胃组织总RNA中克隆福寿螺多功能纤维素酶基因egx序列，连接克隆载体pMD18-T，再通过Eco RⅠ和NotⅠ两个酶切位点分别与表达载体pET-32a（+）及pcDNA3.1（+）连接，构建原核、真核表达载体，并在E.coli BL-21（DE3）和猪肾细胞PK15中进行表达，最后采用DNS法测定表达产物酶学活性。【结果】RT-PCR扩增得到1 326 bp的序列，包括1 185 bp的编码序列和部分侧翼序列，编码序列与已报道的福寿螺多功能纤维素酶基因cDNA序列相似性为99%，氨基酸序列相似性为100%。原核细胞表达产物经包涵体复性后，对羧甲基纤维素钠、2-羟乙基纤维素、羟乙基纤维素、微晶纤维素、木聚糖的水解活性分别为：24.78、15.67、18.42、600.91和175.43 U•mg-1；而在PK15细胞中重组酶对以上5种底物的活性分别为：0.84、0.78、1.01、14.62和4.23 U•mL-1。【结论】克隆出了福寿螺多功能纤维素酶基因，并能在大肠杆菌和哺乳动物细胞中成功地表达。

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

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 EcoRⅠand NotⅠrestriction 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.

Key words: Ampullaria crossean, multi-functional cellulose, prokaryotic expression, eukaryotic expression, emzyme assay

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.

HUANG Miao-Rong, LIU De-Wu, WU Zhen-Fang. Cloning and Functional Expression of a Multi-Functional Cellulase Gene egx from Mollusca, Ampullaria crossean in vitro[J]. Scientia Agricultura Sinica, 2011, 44(17): 3641-3648.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2011.17.017

https://www.chinaagrisci.com/CN/Y2011/V44/I17/3641

参考文献

[1]Lynd L R, Weimer P J, van Zyl W H, Pretorius I S. Microbial cellulose utilization: fundamentals and biotechnology. Microbiology and Molecular Biology Reviews, 2002, 66(3): 506-577.

[2]Yörük M A, Gül M, Hayirli A, Karaoglu M. Multi-enzyme supplementation to peak producing hens fed corn-soybean meal based diets. International Journal of Poultry Science, 2006, 5(4): 374-380.

[3]Benes A, Guenter W, Marquardt R R, Rotter B A. Effect of β-glucanase/pentosanase enzyme supplementation on the performance of chickens and laying hens fed wheat, barly, naked oats and rye diets. Canadian Journal of Animal Science, 1993, 73: 941-951.

[4]Li W F, Feng J, Xu Z R, Yang C M. Effects of non-starch polysaccharides enzymes on pancreatic and small intestinal digestive. World Journal of Gastroenterology, 2004, 10(6): 856-859.

[5]Omogbenigun F O, Nyachoti C M, Slominski B A. Dietary supplementation with multienzyme preparations improves nutrient utilization and growth performance in weaned pigs. Journal of Animal Science, 2004, 82: 1053-1061.

[6]Smith V H, Tilman G D, Kekola J C. Eutrophication: impact of excess nutrient inputs on freshwater, marine, and terrestrial ecosystems. Environmental Pollution, 1999, 100(1-3): 179-196.

[7]Jongbloed A W, Lenis N P. Environmental concerns of animal manure. Journal of Animal Science, 1998, 76: 2641-2648.

[8]Mallin M A, Cahoon L B. Industrialized animal production-a major source of nutrient and microbial pollution to aquatic ecosystems. Population and Environment, 2003, 24(5): 369-385.

[9]Singh A, Hayashi K. Microbial cellulases: protein architecture, molecular properties and biosynthesis. Advances in Applied Microbiology, 1995, 40: 1-44.

[10]Muthuvelayudham R, Viruthagiri T. Fermentative production and kinetics of cellulase protein on trichoderma reesei using sugarcane bagasse and rice straw. African Journal of Biotechnology, 2006, 5 (20): 1873-1881.

[11]Reese E T, Siu R G H, Levinson H S. The biological degradation of soluble cellulose derivatives and its relationship to the mechanism of cellulose hydrolysis. Journal of Bacteriology, 1950, 59: 485-497.

[12]Wang J, Ding M, Li Y H, Chen Q X, Xu G J, Zhao F K. Isolation of a multi-functional endogenous cellulase gene from mollusc, ampullaria crossean. Acta Biochimica Biophysica Sinica, 2003, 35(10): 941-946.

[13]高中华, 许根俊, 赵辅昆. 福寿螺多功能纤维素酶 EGXA 在酿酒酵母中的表达. 浙江理工大学学报，2007, 24(4): 479-482.

Gao Z H, Xu G J, Zhao F K. Expression of a multi-functional endogenous cellulase gene from Mollusc, Ampullaria crossean in Saccharomyces cerevisiae. Journal of Zhejiang Sci-Tech University, 2007, 24(4): 479-482. (in Chinese)

[14]赵颖, 丁明, 高如丽, 许根俊, 赵辅昆. 福寿螺多功能纤维素酶(EGXA)的结构功能研究. 浙江理工大学学报, 2008, 25(5): 535-538.

Zhao Y, Ding M, Gao R L, Xu G J, Zhao F K. Study the function and structure of a multi-functional cellulase from a mollusca , ampullaria crossean. Journal of Zhejiang Sci-Tech University, 2008, 25(5): 535-538. (in Chinese)

[15]Cheng S J, Yang P Z, Guo L Q, Lin J F, Lou N N. Expression of multi-functional cellulase gene mfc in coprinus cinereus under control of different basidiomycete promoters. The Journal of Bioresource Technology, 2009, 100: 4475-4480.

[16]高如丽. 福寿螺多功能纤维素酶EGXA及其结构域在昆虫细胞Tn5中的重组表达与性质分析[D]. 扬州: 扬州大学, 2007.

Gao R L. Enzymatic and molecular properties of recombinant EGXA, a multifunctional cellulase from Mollusca, Ampullaria Crossean expressed in insect cells Tn5[D]. Yangzhou: Yangzhou University, 2007. (in Chinese)

[17]Ghose T K. Measurment of cellulase activities. Pure and Applied Chemistry, 1987, 59(2): 257-268.

[18]Wood T M, Bhat K M. Methods for measuring cellulose activities. Methods Enzymolohy, 1988, 160: 87-112.

[19]Saha B C. Production, purification and properties of xylanase from a newly isolated fusarium proliferatum. Process Biochemistry, 2002, 37: 1279-1284.

[20]Kozak M. An analysis of 5’-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Research, 1987, 15(20): 8125-8148.

[21]Peti W, Page R. Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expression and Purification, 2007, 51: 1-10.

[22]Gong P S, Luo G M. Recent progress in renaturation of inclusion bodies. China Biotechnology, 2003, 23: 73-77.

[23]Xing J L, Yang X M, Yao X Y, Song F, Chen Z N. Prokaryotic expression and renaturation of engineering chimeric fab antibody against human hepatoma. World Journal of Gastroenterol, 2004, 10(14): 2029-2033.

[24]戎晶晶, 刁振宇, 周国华. 大肠杆菌表达系统的研究进展. 药物生物技术, 2005, 12(6): 416-420.

Rong J J, Diao Z Y, Zhou G H. Research progress on E.coli expression system. Pharmaceutical Biotechnology, 2005, 12(6): 416-420. (in Chinese)

[25]王骥. 福寿螺（ampullaria crossean）多功能纤维素酶EGX的研究[D]. 上海: 中国科学院, 2003.

Wang J. The study on the multi-functional cellulase EGX from Mollusca,Ampullaria crossean[D]. Shanghai: Chinese Academy of Sciences, 2003. (in Chinese)

[26]Aricescu A R, Lu W X, Jones E Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr Section D Biological Crystallography, 2006, 62(10): 1243-1250.

[27]Nettleship J E, Assenberg R, Diprose J M, Rahman-Huq N, Owens R J. Recent advances in the production of proteins in insect and mammalian cells for structural biology. Journal of Structural Biology, 2010, 172: 55-65.

[28]Takeshita F, Takase K, Tozuka M, Saha S, Okuda K, Ishii N, Sasaki S. Muscle creatine kinase/SV40 hybrid promoter for muscle-targeted long-term transgene expression. International Journal of Molecular Medicine, 2007, 19: 309-315.

[29]Hershberg R, Petrov D A. Selection on codon bias. The Journal of Annual Review of Genetics, 2008, 42: 287-299.