美洲大蠊内生菌几丁质酶基因的克隆、表达及其活性研究
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摘要
目的:对美洲大蠊内生黏质沙雷氏菌几丁质酶ChiA基因进行克隆、可溶性表达及功能验证。方法:PCR扩增ChiA基因,TA亚克隆,构建重组表达载体ChiA/p ET21b,生物信息学分析和低温诱导表达、SDS-PAGE、Western blot鉴定,打孔法对几丁质酶活性检测。结果:PCR成功扩增了ChiA基因序列,该序列与GenBank上的S. marcescens ChiA基因序列同源性达99%;该序列编码571个氨基酸,并能够在原核系统中稳定表达。可溶性表达分析显示:通过低温诱导表达,获得可溶性的目的蛋白;活性试验发现:含目的蛋白的表达产物可分解几丁质,且活性强于美洲大蠊内生黏质沙雷氏菌。结论:从美洲大蠊内生黏质沙雷氏菌基因组中成功克隆了几丁质酶ChiA基因,通过原核表达系统获得了具有较强活性的可溶性几丁质酶ChiA,为其应用奠定了基础。
Objective: To clone the chitinase gene ChiA from the endophytes of Periplaneta americana soluble expression of the protein and to identify its function. Methods: The chitinase gene ChiA was amplified by PCR from the DNA of Serratia marcescens,which was separated from the gut of Periplaneta americana and obtained by subcloning. The expression plasmid ChiA/pET21 b was constructed and analyzed by bioinformatics. The plasmid was transformed into E. coli BL21( DE3) and the postive strains were induced by IPTG at 20℃ for 20 h. The bioactivity of the protein was determined by small punch test. Results: The cloned sequence was associated with Serratia marcescens ChiA gene of GenBank and their homology was 99%. The sequence encoded a protein containing of 571 amino acids and expressed stably in prokaryotic system. SDS-PAGE/Western blot show that the soluble target protein was obtained. The small punch test suggested that the target protein had the activity of decomposing chitin and was stronger than that of the Serratia marcescens. Conclusion: The chitinase gene ChiA of the Serratia marcescens from the gut of Periplaneta americana was cloned successfully. The soluble chitinase that shows marked bioactivity was attained by prokaryotic expression system,which has provided theoretical basis for its further application.
Objective: To clone the chitinase gene ChiA from the endophytes of Periplaneta americana soluble expression of the protein and to identify its function. Methods: The chitinase gene ChiA was amplified by PCR from the DNA of Serratia marcescens,which was separated from the gut of Periplaneta americana and obtained by subcloning. The expression plasmid ChiA/pET21 b was constructed and analyzed by bioinformatics. The plasmid was transformed into E. coli BL21( DE3) and the postive strains were induced by IPTG at 20℃ for 20 h. The bioactivity of the protein was determined by small punch test. Results: The cloned sequence was associated with Serratia marcescens ChiA gene of GenBank and their homology was 99%. The sequence encoded a protein containing of 571 amino acids and expressed stably in prokaryotic system. SDS-PAGE/Western blot show that the soluble target protein was obtained. The small punch test suggested that the target protein had the activity of decomposing chitin and was stronger than that of the Serratia marcescens. Conclusion: The chitinase gene ChiA of the Serratia marcescens from the gut of Periplaneta americana was cloned successfully. The soluble chitinase that shows marked bioactivity was attained by prokaryotic expression system,which has provided theoretical basis for its further application.
引文
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[11]贺淹才,刘爱花,柴思捷,等.改进RBB染色法初步筛选几丁质酶基因chiA的表达.华侨大学学报(自然科学版),2008,29(4):563-566.He Y C,Liu A H,Chai S J,et al. Improved RBB dyeing method screen bacteria of expression of chintinase chiA. Journal of Huaqiao University(Natural Science),2008,29(4):563-566.
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[13]Francesco M,Sushmita M. Natural computing methods in bioinformatics:A survey. Information Fusion,2009,10(3):211-216.
[14]Balzer S,Kucharova V,Megerle J,et al. A comparative analysis of the properties of regulated promoter systemscommonly used for recombinant gene expression in Escherichia coli. Microb Cell Fact,2013,12(1):1-14.
[15]刘丹,孟凡欢,李爽,等.一株产几丁质酶渤海丝状真菌(Alternaria tenuissima)的筛选及鉴定.中国酿造,2012,31(2):45-28.Liu D,Meng F H,Li S,et al. Screening and identification of chitinase-producing fungus(Alternaria tenuissima)from Bohai Bay. China Brewing,2012,31(2):45-28.
[16]李晶,于丽萍,刘宇帅,等.黏质沙雷氏菌几丁质酶基因的研究进展.基因组学与应用生物学,2017,36(9):3837-3841.Li J,Yu L P,Liu Y S,et al. Research progress of chitinase gene from Serratia marcescens. Genomics and Applied Biology,2017,36(9):3837-3841.
[17]孟利强,沙长青,张先成,等.黏质沙雷氏菌几丁质酶基因ChiA克隆及生物信息学分析.微生物学杂志,2016,36(1):62-68.Meng L Q, Sha C Q, Zhang X C, et al. Cloning and bioinformatics analysis of chitinase gene ChiA from Serratia marcescens. Journal of Microbiology,2016,36(1):62-68.
[18] Shapira R.,Ordentlich A.,Chet I,et al. Control of plant diseases by chitinase expressed from cloned DNA in Escherichia coli. Phytopathology,1989,79(11):1246-1249.
[2]Dutta J,Tripathi S,Dutta P K,et al. Progress in antimicrobial activities of chitin,chitosan and its oligosaccharides:a systematic study needs for food applications. Food Science and Technology International,2012,18(1):3-34.
[3]Horn S J,Sikorski P,Cederkvist J B,et al. Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides. P Natl Acad Sci USA,2006,103(48):18089-18094.
[4]李海浪.壳聚糖衍生物的制备及其在药物载体中的应用研究.上海:中国科学院大学,2014.Li H L. Study on the preparation of chitosan derivatives and their applications in drug delivery. Shanghai:University of Chinese Academy of Sciences,2014.
[5] Benecke U. Bacillus chitinovorous einen chitin zersetzenden Spaltpilz. Bot Ztg,1905,63(8):227-242.
[6]Merzendorfer H. The cellular basis of chitin synthesis in fungi and insect:common principles and differaices. Eur J Cell Biol.2011,90(9):759-769.
[7]糜艳霞,任慧,张常,等.几丁质酶的研究进展.生命科学研究,2015,19(5):437-442.Mi Y X, Ren H, Zhang C, et al. Advances in study and application on chitinase. Life Science Research,2015,19(5):437-442.
[8]白腾飞,刘月芹.沙雷氏菌抗生性次级代谢产物合成机制.微生物学杂志,2017,37(4):115-119.Bai T F,Liu Y Q. A Survey of synthesis mechanism of antibiotic secondary metabolites by Serratia spp.. Journal of Microbiology,2017,37(4):115-119.
[9]方霞,沈娟,王影姣,等.药用昆虫美洲大蠊内生放线菌的分离和鉴定.中国病原生物学杂志,2016,11(6):550-565.Fang X,Shen J,Wang Y J,et al. Isolation and identification of endophytic actinomycetes from medical insects(Periplaneta Americana). Journal of Pathogen Biology,2016,11(6):550-565.
[10] Van Arnam E B,Currie C R,Clardy J. Defense contracts:molecular protection in insect-microbe symbioses. Chemical Society Reviews,2018,47(57):1638-1651.
[11]贺淹才,刘爱花,柴思捷,等.改进RBB染色法初步筛选几丁质酶基因chiA的表达.华侨大学学报(自然科学版),2008,29(4):563-566.He Y C,Liu A H,Chai S J,et al. Improved RBB dyeing method screen bacteria of expression of chintinase chiA. Journal of Huaqiao University(Natural Science),2008,29(4):563-566.
[12] Chung W C,Chen L L,Lo W S,et al. Complete genome sequence of Serratia marcescens WW4. Genome Announc,2013,4(2):12-13.
[13]Francesco M,Sushmita M. Natural computing methods in bioinformatics:A survey. Information Fusion,2009,10(3):211-216.
[14]Balzer S,Kucharova V,Megerle J,et al. A comparative analysis of the properties of regulated promoter systemscommonly used for recombinant gene expression in Escherichia coli. Microb Cell Fact,2013,12(1):1-14.
[15]刘丹,孟凡欢,李爽,等.一株产几丁质酶渤海丝状真菌(Alternaria tenuissima)的筛选及鉴定.中国酿造,2012,31(2):45-28.Liu D,Meng F H,Li S,et al. Screening and identification of chitinase-producing fungus(Alternaria tenuissima)from Bohai Bay. China Brewing,2012,31(2):45-28.
[16]李晶,于丽萍,刘宇帅,等.黏质沙雷氏菌几丁质酶基因的研究进展.基因组学与应用生物学,2017,36(9):3837-3841.Li J,Yu L P,Liu Y S,et al. Research progress of chitinase gene from Serratia marcescens. Genomics and Applied Biology,2017,36(9):3837-3841.
[17]孟利强,沙长青,张先成,等.黏质沙雷氏菌几丁质酶基因ChiA克隆及生物信息学分析.微生物学杂志,2016,36(1):62-68.Meng L Q, Sha C Q, Zhang X C, et al. Cloning and bioinformatics analysis of chitinase gene ChiA from Serratia marcescens. Journal of Microbiology,2016,36(1):62-68.
[18] Shapira R.,Ordentlich A.,Chet I,et al. Control of plant diseases by chitinase expressed from cloned DNA in Escherichia coli. Phytopathology,1989,79(11):1246-1249.
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