鸭疫里默氏杆菌CAMP基因的克隆、原核表达及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2021.12.003

Abstract

Abstract: The aim of this study was to clone the cooperative hemolysin protein (CAMP) gene of Riemerella anatipestifer, and analyze its prokaryotic expression and bioinformatics. CAMP gene was amplified and cloned from RA Guizhou serotype 2 strain(RA-SS-8), and the recombinant prokaryotic expression plasmid pET-28a-CAMP was constructed.The plasmid was transformed into Escherichia coli Rosetta(DE3) competent cells.After identification, the recombinant bacteria were induced to express and purified with IPTG.SDS-PAGE and Western blotting were used to analyze the characteristics of the expressed protein.CAMP gene was analyzed by bioinformatics analysis software.The results showed that the size of CAMP gene was 1 026 bp, and the sequence of CAMP gene was 99.7% simarility with 10 RA reference strains published in GenBank, such as RA-LZ01 (CP045564.1).By BamH Ⅰ and Xho Ⅰ double digestion, the size of 5 369 and 1 026 bp fragments were obtained, indicating that the pET-28a-CAMP recombinant plasmid was successfully constructed.SDS-PAGE and Western blotting analysis results showed that the expressed recombinant protein was about 37 ku in soluble form, and could react with His-tag monoclonal antibody at 37 ku, which was consistent with the expected results.Bioinformatics analysis results showed that the molecular formula of CAMP was C₁₆₇₀H₂₆₅₉N₄₃₇O₅₀₀S₁₂, containing 341 amino acids, the theoretical isoelectric point (pI) was 5.62, and the coefficient of instability was 40.71, indicating that CAMP was an unstable and weakly acidic protein.The results of hydrophobicity prediction showed that CAMP protein was hydrophilic.CAMP protein had no transmembrane domain, no signal peptide, 3 O-glycoylation sites, no N-glycoylation sites, 34 phosphorylation sites and a total of 17 epitopes.The secondary and tertiary structures showed that the protein was dominated by alpha helix and random coil.The above results might provide reference for further study on the function of CAMP protein and the development of Riemerella anatipestifer vaccine.

Key words: Riemerella anatipestifer; CAMP gene; prokaryotic expression; bioinformatics analysis

CLC Number:

MEI Shihui, CHEN Guoquan, YAN Chaohua, WANG Na, ZHOU Bijun, CHENG Zhentao, WANG Kaigong, WEN Ming. Cloning, Prokaryotic Expression and Bioinformatics Analysis of CAMP Gene of Riemerella anatipestifer[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4348-4360.

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Cloning, Prokaryotic Expression and Bioinformatics Analysis of CAMP Gene of Riemerella anatipestifer

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