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中国精品科技期刊2020
胡金双, 黄燕燕, 陈思敏, 邝嘉华, 赵珊, 彭鑫, 刘冬梅. 蜡样芽孢杆菌LJ01中亚硝酸盐还原酶的基因克隆和生物信息学分析[J]. 食品工业科技, 2020, 41(6): 94-98,105. DOI: 10.13386/j.issn1002-0306.2020.06.016
引用本文: 胡金双, 黄燕燕, 陈思敏, 邝嘉华, 赵珊, 彭鑫, 刘冬梅. 蜡样芽孢杆菌LJ01中亚硝酸盐还原酶的基因克隆和生物信息学分析[J]. 食品工业科技, 2020, 41(6): 94-98,105. DOI: 10.13386/j.issn1002-0306.2020.06.016
HU Jin-shuang, HUANG Yan-yan, CHEN Si-min, KUANG Jia-hua, ZHAO Shan, PENG Xin, LIU Dong-mei. Gene Cloning and Bioinformatics Analysis of Nitrite Reductase from Bacillus cereus LJ01[J]. Science and Technology of Food Industry, 2020, 41(6): 94-98,105. DOI: 10.13386/j.issn1002-0306.2020.06.016
Citation: HU Jin-shuang, HUANG Yan-yan, CHEN Si-min, KUANG Jia-hua, ZHAO Shan, PENG Xin, LIU Dong-mei. Gene Cloning and Bioinformatics Analysis of Nitrite Reductase from Bacillus cereus LJ01[J]. Science and Technology of Food Industry, 2020, 41(6): 94-98,105. DOI: 10.13386/j.issn1002-0306.2020.06.016

蜡样芽孢杆菌LJ01中亚硝酸盐还原酶的基因克隆和生物信息学分析

Gene Cloning and Bioinformatics Analysis of Nitrite Reductase from Bacillus cereus LJ01

  • 摘要: 为研究蜡样芽孢杆菌LJ01(Bacillus cereus LJ01)中亚硝酸盐还原酶(NiR)的酶学性质,获得高表达量的基因工程菌,本文对B.cereus LJ01的NiR序列进行了生物信息学分析,并将NiR基因克隆到表达载体pET-28a(+)和pET-32a(+)中,构建了基因工程菌pET-28a(+)-nir-BL21和pET-32a(+)-nir-BL21,随后探究了不同诱导条件对重组NiR表达量的影响。结果表明NiR编码蛋白的理论分子量约为60 kDa,理论pI为5.47,二级结构主要为α-螺旋和无规则卷曲,是不具有跨膜结构的亲水性蛋白。随着诱导温度的升高,重组NiR的表达量逐渐减少,诱导温度为16℃时重组NiR表达量最高。在同一诱导温度下,NiR在pET-28a(+)-nir-BL21中的表达量明显高于pET-32a(+)-nir-BL21,因此选用pET-28a(+)-nir-BL21作为基因工程菌。从B.cereus LJ01中克隆了NiR基因,构建了基因工程菌pET-28a(+)-nir-BL21,为该重组NiR的理化性质研究和食源芽孢杆菌中NiR的异源表达奠定了基础。

     

    Abstract: To study the enzymatic properties of nitrite reductase(NiR)in Bacillus cereus LJ01 and obtain highly expressed genetic engineering bacteria,the NiR sequence of B. cereus LJ01 was analyzed by bioinformatics. Then the NiR gene was cloned into expression vectors pET-28a(+)and pET-32a(+),and the genetic engineering bacteria pET-28a(+)-nir-BL21 and pET-32a(+)-nir-BL21 were constructed. Afterwards,the effects of different induction temperatures on the expression of recombinant NiR was studied. The results showed that the theoretical molecular weight of the protein encoded by NiR was about 60 kDa,the theoretical pI was 5.47,the secondary structure was mainly alpha-helix and irregular curl,and it was a hydrophilic protein without transmembrane structure. With the increasing of induction temperature,the expression of recombinant NiR decreased gradually,and the highest expression of recombinant NiR was found at the induction temperature of 16℃. At the same induction temperature,the expression of NiR in pET-28a(+)-nir-BL21 was significantly higher than that in pET-32a(+)-nir-BL21.Therefore,pET-28a(+)-nir-BL21 was selected as the genetic engineering bacteria. This study cloned NiR from obtained B. cereus LJ01 and obtained pET-28a(+)-nir-BL21 as engineering bacteria,which laid a foundation for the study of the physical and chemical properties of the recombinant NiR and the heterologous expression of NiR in foodborne Bacillus spp.

     

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