We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
副结核分枝杆菌胞外 GDSL 脂肪酶 MAP_2739 的 酶学特性研究.
- Authors
王建国; 陈凡若; 鹿萍; 臧鑫鑫; 党光辉; 美艳; 崔宁; 冯婷婷; 张嘉俊; 王慧; 崔莹莹; 崔子寅; 刘思国
- Abstract
In the early stage of this study, an unreported GDSL lipase family protein of Mycobacterium avium subsp. paratuberculosis (MAP), MAP_ 2739 was discovered. To study the enzymatic properties of MAP_ 2739, bioinformatics websites and software were used to analyze the conserved domain, secondary structure and 3D homology modeling ofMAP_2739, and further confirmed that it belonged to the GDSL lipase family. The target gene of MAP_ 2739 was obtained by PCR amplification of about 891bp using the genomic DNA of MAP reference strain as a template, ligated to the expression vector pET-22b, verified by sequencing, and successfully constructed a recombinant expression strain of E. coli, which was purified by IPTG-induced expression, protein refolding, and affinity chromatography. The expression and purification of the target protein were analyzed by SDS-PAGE and verified by western blot. The results showed that the recombinant purified MAP_2739 protein (rMAP _2739) with inclusion body expression was successfully obtained. The rabbit polyclonal antibodies against this protein were prepared, and the serum titer was 1 :51200 determined by ELISA. The MAP reference strain fraction was isolated by differential centrifugation, and the target bands appeared at 35ku in the cell wall, cell membrane and cell pulp, as verified by western blot using this polyclonal antibody as primary antibody, indicating that MAP _2739 is an extracellular protein. The enzymatic activity was determined by using p-nitrophenyl esters (p-NPs) as substrate, and the validation proved that MAP_ 2739 protein was lipase, which could hydrolyze long-chain fatty acids with the optimal enzymatic reaction pH value at 9.0, and rMAP _2739 protein still had lipase activity after pretreatment by pasteurization. The recombinant plasmid pET-22b-map_2739 gene was used as template, amplified by PCR using site-directed mutagenesis primers, verified by sequencing, transformed into E. coli expression strain, purified by western blot to verify protein expression, and analyzed by enzyme activity assay, respectively. The results showed that the purified proteins were successfully obtained for each point mutation, and S46 and D204 were identified as the enzyme active sites of MAP _2739. To sum up, this study demonstrated for the first time that MAP_ 2739 is a MAP extracellular GDSL lipase, which laid a foundation for further study on the pathogenesis of MAP.
- Subjects
MYCOBACTERIUM avium paratuberculosis; BINDING sites; ESCHERICHIA coli; GENE expression; SITE-specific mutagenesis; TITERS
- Publication
Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao, 2023, Vol 45, Issue 4, p331
- ISSN
1008-0589
- Publication type
Article
- DOI
10.3969/j.issn.1008-0589.202211004