苏云金芽孢杆菌（Bacillus thuringiensis）几丁质酶基因的克隆、表达及其工程菌的构建
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摘要
从本室保存的Bacillus thuringiensis(Bt)菌株中，筛选出几丁质酶高产菌株WB-7。根据已经在GenBank上登录的Bt几丁质酶基因序列设计1对引物QCL1/QCL2，用PCR方法扩增出的几丁质酶全长基因，将PCR产物直接连接到pMD-18T克隆载体上转入E. coli DH5α中克隆得到2025bp包含氨基酸全序列的几丁质酶基因，并命名为Chi7，在GenBank上的登录注册号为AY074882。利用Blast软件进行该基因的同源性分析，结果表明：chi7基因与已知的Bt和Bc几丁质酶基因都有90％以上的同源性；而与其它细菌的几丁质酶基因同源性不高，如与Bacillus subtilis、Bacillus circulans的几丁质酶基因的同源性分别为50％、52％的。将chi7基因序列翻译为Chi7蛋白质氨基酸序列，该基因编码674个氨基酸残基。利用蛋白质分析软件对Chi7蛋白进行功能结构和空间结构分析，结果表明：经在线的Smart软件进行氨基酸的序列分析，推导出的Chi7蛋白质一级结构功能域包含有：7-26信号肽区、chiA几丁质酶催化区、485-557类纤连蛋白Ⅲ型区、579-672几丁质结合区4个结构功能域；经跨膜区分析在线软件TMHMM2.0分析，表明Chi7蛋白前端的信号肽是跨膜信号，同时SignalP分析也表明：Chi7蛋白的成熟加工剪切位点在第32-33的Ala-Asp间。用在线蛋白质分析软件ExPASy Proteomics tools进行chi7蛋白结构分析，结果表明：在氨基酸残基200-300间有可能存在coiled coil区域，整个蛋白质中没有亮氨酸拉链结构；在蛋白的二级结构中有19.56％的螺旋结构、25.78％的折叠结构、54.67％的成环结构；整个蛋白质的三维结构呈紧密的球状。构建pBluescript SK原核表达载体在E.coli中检测到Bt几丁质酶活性，但是表达的Bt几丁质酶活性不高。
将Bt chi7基因连接到枯草芽孢杆菌(Bs)表达载体pUS186，电激转化到Bs菌株QB1098中筛选重组质粒pUS-chi7。将重组质粒pUS-chi7电激转化到Bs受体菌株BS-2中，用PCR检测重组质粒pUS-chi7是否导入，最后筛选得到5株含有Bt几丁质酶基因BS-2工程菌。
The chitinase gene named chi7(2025bp) from Bacillus thuringiensis strain WB7 with high chitinase-producing ability was cloned in Escherichia coli DH5 a with pMD-18T cloning vector by the PCR method. The GenBank accession number ofchil was AY074882. With the software of Blast online, the sequence analysis showed that more than 90% high degree of identity with other BT chitinase gene and Bacillus cereus chitinase gene; moreover, it showed lower identity with other Bacillus chitinases gene, such as B. circulans and B. subtilis, 52% and 50% respectively. The amino acid sequence of Chi7 chitinase was also deduced. The functional and spacial structure was analysized. With the help of SMART software online for functional analysis, the sequence of 674 amino acids includes a signal peptide(7-26), a ChiA chitin catalytic domain, a fibronectin-like domain (485-557), and a chitin-binding domain(579-672). With the signal peptide analysis software TMHMM2.0, the preceding peptide of Chi7 was a transmembrane peptide, the ra
w cleavage site of Chi7 chitinase was between Ala-Asp at 32-33 with the SignalP analysis. With the help of ExPASy Proteomics tools, the spacial structure analysis showed that it may exist a coiled coil region at amino acid residue 200-300; there was no leucine zipper structure in the whole amino acid sequence; with PredictProtein software online of Colombia University for structure analysis, it formed 19.56% helix, 25.78% sheet, and 54.67% loop in secondary structure of Chi7 chitinase; the whole Chi7 protein appeared as compact, as a globular domain in tertiary structure. An expression vector
pBluescript SK was constructed and detected BT chitinase activity was detected in E. coli, but the chitinase activity was not high. The Bt chil gene was ligated with the plasmid pUS186, a kind of Bacillus subtilis expression plasmid, then transformed to B. subtilis strain QB1098 by electroporation. The recombinant plasmid pUS-chil was also ' transformed to B. subtilis strain BS-2, detected by PCR method. Therefore, 5 engineering bacteria strains of B. subtilis with BT chitinase gene were obtained and kept.
将Bt chi7基因连接到枯草芽孢杆菌(Bs)表达载体pUS186，电激转化到Bs菌株QB1098中筛选重组质粒pUS-chi7。将重组质粒pUS-chi7电激转化到Bs受体菌株BS-2中，用PCR检测重组质粒pUS-chi7是否导入，最后筛选得到5株含有Bt几丁质酶基因BS-2工程菌。
The chitinase gene named chi7(2025bp) from Bacillus thuringiensis strain WB7 with high chitinase-producing ability was cloned in Escherichia coli DH5 a with pMD-18T cloning vector by the PCR method. The GenBank accession number ofchil was AY074882. With the software of Blast online, the sequence analysis showed that more than 90% high degree of identity with other BT chitinase gene and Bacillus cereus chitinase gene; moreover, it showed lower identity with other Bacillus chitinases gene, such as B. circulans and B. subtilis, 52% and 50% respectively. The amino acid sequence of Chi7 chitinase was also deduced. The functional and spacial structure was analysized. With the help of SMART software online for functional analysis, the sequence of 674 amino acids includes a signal peptide(7-26), a ChiA chitin catalytic domain, a fibronectin-like domain (485-557), and a chitin-binding domain(579-672). With the signal peptide analysis software TMHMM2.0, the preceding peptide of Chi7 was a transmembrane peptide, the ra
w cleavage site of Chi7 chitinase was between Ala-Asp at 32-33 with the SignalP analysis. With the help of ExPASy Proteomics tools, the spacial structure analysis showed that it may exist a coiled coil region at amino acid residue 200-300; there was no leucine zipper structure in the whole amino acid sequence; with PredictProtein software online of Colombia University for structure analysis, it formed 19.56% helix, 25.78% sheet, and 54.67% loop in secondary structure of Chi7 chitinase; the whole Chi7 protein appeared as compact, as a globular domain in tertiary structure. An expression vector
pBluescript SK was constructed and detected BT chitinase activity was detected in E. coli, but the chitinase activity was not high. The Bt chil gene was ligated with the plasmid pUS186, a kind of Bacillus subtilis expression plasmid, then transformed to B. subtilis strain QB1098 by electroporation. The recombinant plasmid pUS-chil was also ' transformed to B. subtilis strain BS-2, detected by PCR method. Therefore, 5 engineering bacteria strains of B. subtilis with BT chitinase gene were obtained and kept.
引文
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[5]喻子牛．苏云金杆菌制剂的生产和应用．北京：农业出版社，1993．
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