北京林业大学学报2024，Vol.46Issue(4)：84-90,7.DOI:10.12171/j.1000-1522.20220231
蜡样芽孢杆菌NJSZ-13中蛋白酶ATP-α与ClpX的杀线虫活性研究
Prokaryotic expression of nematicidal proteases ATP-α and ClpX from Bacillus cereus NJSZ-13
摘要
Abstract
[Objective]In order to elucidate the molecular mechanism of nematicidal proteases ATP-α and ClpX in Bacillus cereus NJSZ-13,prokaryotic expression vectors of nematicidal proteases ATP-α and ClpX were constructed.[Method]The nematicidase-encoding genes atpA and clpX were cloned,PCR-amplified the nematicidase-encoding gene by means of genetic engineering and connected with the vector pET-21b to construct a recombinant vector.The recombinant plasmid was extracted and transferred into the protein expression vector E.coli BL21(DE3).The transformation effect was verified by colony PCR and sequencing.IPTG was added to induce protein expression,and the recombinant protein was purified using a Ni-NTA column.The purification effect of ATP-α and ClpX was verified by SDS-PAGE and Western blot,and the nematicidal activity was determined.[Result]Colony PCR verification and sequencing showed that the recombinant vector contained protease-encoding genes atpA and clpX,and the gene sequence was consistent with the reference sequence,proving that the target genes had been successfully inserted into the BL21(DE3)expression vector.SDS-PAGE and Western blot showed that the recombinant protein was correctly induced and purified,and the prokaryotic expression vectors of nematicide protease ATP-α and ClpX were successfully constructed.The results of nematicidal activity assay showed that both ATP-α and ClpX had strong nematicidal effects.ATP-α reached a nematicidal rate of 66.75%at 72 h,and ClpX reached a nematicidal rate of 75.46%at 72 h.At the same time,the ability of the two proteases working together to kill the nematodes was greatly enhanced,showing that the nematicidal rate of 66.32%in 24 h and 91.01%in 72 h.[Conclusion]ATP-α and ClpX have higher nematicidal activity after prokaryotic expression and purification,and the two together have stronger nematicidal effect on B.xylophilus.It is proved that protease ATP-α and ClpX are important killing factors,which provides new clues and basis for the design and screening of killing drugs.关键词
蜡样芽孢杆菌NJSZ-13/松材线虫/ATP-α/ClpX/原核表达Key words
Bacillus cereus NJSZ-13/Bursaphelenchus xylophilus/ATP-α/ClpX/prokaryotic expression分类
农业科技引用本文复制引用
孙玉凤,谈家金,袁裕超,赵晓佳,叶建仁..蜡样芽孢杆菌NJSZ-13中蛋白酶ATP-α与ClpX的杀线虫活性研究[J].北京林业大学学报,2024,46(4):84-90,7.基金项目
国家重点研发计划(2021YFD1400900). （2021YFD1400900）
