羊源多杀性巴氏杆菌sodA基因的克隆、表达及其生物信息学分析

doi:10.16431/j.cnki.1671-7236.2018.08.004

Abstract

Abstract:

To investigate the biological functions of superoxide dismutase (SOD) of Pasteurella multocida,the sodA gene was cloned,expressed and analyzed by phylogenetic and bioinformatics,respectively.A pair of primers was designed based on the sodA gene sequence of Pasteurella multocida HN06 strain in GenBank.The PCR products were ligated with pET-28a(+) vector to construct pET-28a(+)-sodA recombinant plasmids.The recombinant plasmids were transformed into E.coli DH5α competent cells for cloning and then transformed them into E.coli BL21(DE3) competent cells for expression.The expressed proteins were induced by IPTG and identified by SDS-PAGE and Western blotting.The results showed that the target fragment with the length of 645 bp was successfully amplified and the target protein which was 28 ku was successfully expressed.Phylogenetic analysis showed that the gene was closely related to the HN07 (GenBank accession No.:CP007040.1) and Pm70 (GenBank accession No.:AE004439.1) strains.Bioinformatics analysis results of the recombinant protein showed that it was a stable acidic hydrophilic soluble fusion protein.The molecular formula and molecular mass were C₁₀₈₅H₁₆₅₁N₂₉₃O₃₀₉S₉ and 24 032.36 u;The theoretical isoelectric point,extinction coefficient and instability coefficient were 6.19,45 170 and 26.87 (<40),repectively;The half-life of reticulocytes in mammals was estimated to be 30 h;The aliphatic index and total average hydrophobicity (GRAVY) were 82.15 and -0.282,respectively;And the secondary structure was mainly composed of α-helix and random coil.The above results provided references for the further study of the survival mechanism of Pasteurella multocida and the development of a vaccine against pasteurellosis.

Key words: Pasteurella multocida; sodA gene; cloning; prokaryotic expression; evolutionary analysis; bioinformatics analysis

CLC Number:

S852.61

AN Qi, HUANG Haifeng, ZHANG Zhenxing, LI Baobao, ZHANG Mengmeng, ZHENG Yiying, WANG Chengqiang, ZHANG Luyin, YANG Xiaojian, CAO Ruiyong, NIE Xin, DU Li, WANG Fengyang. Cloning, Expression and Bioinformatics Analysis of sodA Gene of Pasteurella multocida[J]. , 2018, 45(8): 2067-2075.

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Cloning, Expression and Bioinformatics Analysis of sodA Gene of Pasteurella multocida

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