羊源多杀性巴氏杆菌ompW基因的克隆及生物信息学分析

试验旨在克隆羊源多杀性巴氏杆菌ompW基因，并对其序列进行生物信息学分析。根据GenBank中多杀性巴氏杆菌HN07株ompW基因序列（登录号：CP007040.1），使用DNAMAN 5.0软件设计1对引物，选取高保真酶PrimeSTAR Max DNA Polymerase进行PCR反应获取目的基因片段，并对ompW基因的核苷酸序列及预测的氨基酸序列进行生物信息学分析。结果表明，PCR扩增产物约为615 bp，编码204个氨基酸。核苷酸同源性比对分析显示，羊源多杀性巴氏杆菌ompW基因与猪源、牛源、禽源多杀性巴氏杆菌同源性较高，而与兔源同源性较低。系统进化树结果发现，羊源多杀性巴氏杆菌ompW基因与猪源多杀性巴氏杆菌ompW基因亲缘关系最近。经生物信息学分析发现，ompW蛋白分子式为C₁₀₀₇H₁₅₆₇N₂₅₇O₂₈₃S₃，分子质量为21.90 ku，理论等电点（pI）为9.16，属碱性蛋白质，疏水指数为96.57，总平均疏水性（GRAVY）为0.173（> 0），属于疏水类蛋白；前21位氨基酸为信号肽，第5－27位氨基酸区域存在1个跨膜区，存在N-糖基化位点及磷酸化位点，不存在O-糖基化位点，具有多个B细胞、CTL细胞及Th细胞抗原表位；二级结构的α-螺旋、延伸链、β-转角和无规则卷曲分别占17.65%、35.29%、3.92%和43.14%；三级结构是呈β-桶状的单聚体，隶属于外膜蛋白家族成员之一。本研究结果为进一步阐明羊源多杀性巴氏杆菌侵染宿主过程中自身的抗宿主免疫胁迫机制及疫苗的开发与研制提供了理论依据。

Cloning and Bioinformatics Analysis of ompW Gene from Pasteurella multocida in Goat

This study was aimed to clone ompW gene of Pasteurella multocida in goat and carry out bioinformatics analysis.According to the ompW gene sequence of Pasteurella multocida strain HN07 (accession No.:CP007040.1) in GenBank,one pair of primers were designed using DNAMAN 5.0 software,and the high-fidelity enzyme PrimeSTAR^® Max DNA Polymerase was selected for PCR reaction to obtained the target gene fragment,the nucleotide and amino acid sequences of ompW gene were analyzed by bioinformatics softwares.The results showed that the length of PCR product was about 615 bp,which encoded 204 amino acids.The nucleotide homology analysis result showed that ompW gene of Pasteurella multocida in goat was highly homologous with porcine,bovine and avian-derived Pasteurella multocida,but not highly homologous to Pasteurella multocida in rabbit.The phylogenetic tree result showed that the relationship was the closest between ompW gene of Pasteurella multocida in goat and porcine.The bioinformatics analysis results showed that the molecular formula of ompW protein was C₁₀₀₇H₁₅₆₇N₂₅₇O₂₈₃S₃,the molecular weight was 21.90 ku,the theoretical isoelectric point (pI) was 9.16,which was a basic protein with a hydrophobic index of 96.57 and a total average hydrophobicity (GRAVY) of 0.173 (> 0),it belonged to a hydrophobic protein.The first 21 amino acids were signal peptides,the 5th to 27th amino acid regions had a transmembrane region,there were N-glycosylation and phosphorylation sites,no O-glycosylation site,and there were multiple B cell,CTL cell and Th cell epitopes.The alpha helix,extended chain,beta turn and random coil of the secondary structure accounts for 17.65%,35.29%,3.92% and 43.14%,respectively.The tertiary structure was a beta barrel monomer belonging to one of the members of the outer membrane protein family.These results provided a theoretical basis for further elucidating the mechanism of anti-host immune stress and the development of vaccines in the process of infecting host of Pasteurella multocida.