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.