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本研究对枯草芽孢杆菌中性蛋白酶的基因进行克隆,并在大肠杆菌中诱导表达。从枯草芽孢杆菌基因组克隆到编码中性蛋白酶的全长基因,构建大肠杆菌原核表达载体pET30b,转化大肠杆菌BL21得到重组工程菌。通过菌落PCR和酶切筛选验证重组体。在25℃,110 r/min条件下,用IPTG诱导重组蛋白表达。表达蛋白用SDS-PAGE验证,并对酶活力进行测定。测序结果表明克隆序列与NCBI上的登录的序列同源性高达99%。SDS-PAGE结果表明诱导出的蛋白相对分子质量56.6 kD,酶活力达到39 U/mL。证明成功克隆得到枯草芽孢杆菌中性蛋白酶基因,并在大肠杆菌中得到高效表达。
Abstract：The study cloned the gene of neutral protease from Bacillus subtilis BS8 and induced expression in E.coli BL21. The full-length gene fragment encoding neutral protease was cloned from the genome of Bacillus subtilis BS8 to construct the prokaryotic expression vector p ET30 b of E. coli and transformed E. coli BL21 to obtain the recombinant engineering bacteria. After that, the transformants were screened and identified by PCR and restriction endonuclease digestion analysis. The recombinant bacteria was induced to express by IPTG in the condition of25℃,110 r/min. The expressing recombinant protein and neutral protease enzyme activity were verified and measured by SDS-PAGE and Folin-phenol method, respectively. DNA sequencing results showed that the homology between cloning sequence and login sequence in NCBI was 99%. SDS-PAGE analysis displayed that the molecular weight of the expressed recombinant products was approximately 56.6 kD with the enzyme activity 39 U/ml, indicating the successful cloning of neutral protease from Bacillus subtilis and the high efficient expression in E. coli.
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基本信息:
DOI：10.13417/j.gab.036.000906
中图分类号:Q78;Q936
引用信息:
[1]王东,钟涛,孙荣,等.枯草芽孢杆菌中性蛋白酶基因的克隆与表达[J].基因组学与应用生物学,2017,36(03):906-910.DOI:10.13417/j.gab.036.000906.
基金信息:
四川省科技厅支撑计划项目(2013GZX0160)资助
2017-03-25
2017-03-25