Expression and purification of Bacillus anthracis phage endolysin PlyL and its role in Bacillus anthracis detection
10.13200/j.cnki.cjb.004661
- VernacularTitle:炭疽芽孢杆菌噬菌体内溶素PlyL的表达、纯化及其在炭疽芽孢杆菌检测中的作用
- Author:
Minghan XU
1
Author Information
1. College of Life Sciences, Jilin Agricultural University, Changchun 130118, Jilin Province, China Corresponding authors: HAN Peng, E⁃mail: hanpeng@jlau.edu.cn
- Publication Type:Journal Article
- Keywords:
Bacillus anthracis;
Endolysin PlyL;
Prokaryotic expression;
ELISA
- From:
Chinese Journal of Biologicals
2026;39(03):282-288
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo express and purify the endolysin PlyL from the Bacillus anthracis prophage LambdaBa02 in prokaryotic cells, and to explore its role in the detection of Bacillus anthracis, so as to provide a novel strategy for the specific and rapid detection of Bacillus anthracis.MethodsAccording to the sequence of Bacillus anthracis vaccine strain CVCC40205 registered in GenBank, the plyL gene was amplified by PCR with plyL gene-specific primers and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant expression plasmid pET28a(+)-His-plyL, which was then transformed into E.coli BL21(DE3) and induced by IPTG. The induced expression conditions were optimized to enhance soluble expression, and the recombinant PlyL protein was purified via nickel-affinity chromatography. The lytic activity of recombinant PlyL protein was assessed by measuring absorbance values, and an ELISA method was established to evaluate the sensitivity and specificity of Bacillus anthracis detection.ResultsThe recombinant expression plasmid pET28a(+)-His-plyL was confirmed to be correctly constructed by NdeⅠ and XhoⅠ double digestion and sequencing. The optimal induction conditions were 0. 5 mmol/L IPTG at 16 ℃ for 16 h. The relative molecular mass of the expressed recombinant PlyL protein was about 28 600. After purification and concentration, the final concentration of the protein was2. 55 mg/mL, and the purity reached 80. 6%. The purified recombinant PlyL protein could reduce the A_(600)value of Bacillus anthracis culture solution. The sensitivity of the preliminarily established ELISA method for detecting Bacillus anthracis was1 × 10~3 CFU/mL, and there was no cross-reactivity against closely related strains such as Bacillus cereus and Bacillus thuringiensis(P < 0. 000 1).ConclusionThe recombinant PlyL protein was successfully expressed and purified in prokaryotic cells, which exhibits both efficient lytic activity and species specificity, making it a promising novel tool for the rapid detection of Bacillus anthracis.
