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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (1): 31-37    DOI: 10.13523/j.cb.20190104
研究报告     
美洲大蠊内生菌几丁质酶基因的克隆、表达及其活性研究 *
许敏华,张晶晶,金小宝,李小波,王艳,马艳()
广东药科大学 广东省生物活性药物研究重点实验室 广州 510006
Cloning\Expression and Bioactivity of the Chitinase Gene ChiA from the Endophytes of Periplaneta americana
Min-hua XU,Jing-jing ZHANG,Xiao-bao JIN,Xia-bo LI,Yan WANG,Yan MA()
Guangdong Pharmaceutical University, Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangzhou 510006, China
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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/pET21b,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。
关键词： 美洲大蠊;  黏质沙雷氏菌;  几丁质酶ChiA;  可溶性表达;  活性验证    
Abstract:

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/pET21b 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 20h. 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.
Key words: Periplaneta americana    Serratia marcescens    Chitinase ChiA    Soluble expression    Bioactivity
收稿日期: 2018-07-25 出版日期: 2019-02-28
ZTFLH:  Q78  
基金资助: * 国家自然科学基金(81502520);广东省自然科学基金(2016A030310299);广东省医学科研基金资助项目(A2016077)
通讯作者: 马艳     E-mail: mayan820901@126.com
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许敏华
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引用本文:

许敏华,张晶晶,金小宝,李小波,王艳,马艳. 美洲大蠊内生菌几丁质酶基因的克隆、表达及其活性研究 *[J]. 中国生物工程杂志, 2019, 39(1): 31-37.

Min-hua XU,Jing-jing ZHANG,Xiao-bao JIN,Xia-bo LI,Yan WANG,Yan MA. Cloning\Expression and Bioactivity of the Chitinase Gene ChiA from the Endophytes of Periplaneta americana. China Biotechnology, 2019, 39(1): 31-37.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190104    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I1/31

图1  ChiA基因的PCR扩增及TA克隆鉴定
图2  ChiA/pET21b表达载体的鉴定
图3  氨基酸序列
图4  亲水性/疏水性预测结果
图5  SDS-PAGE和Western blot结果
图6  活性测试
项目 有无几丁质
酶活性表达		 透明圈大小
(mm)
ChiA/pET21b/BL21上清液 + 24±2.7
WA12 1-18 + 22±2.1
ChiA/pET21b/BL21 + 20±1.6
缓冲液 - ---
表1  几丁质酶活性检测结果汇总表
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