猪胸膜肺炎放线杆菌细胞分裂相关基因的克隆和分析

发布时间：2018-04-19 05:42

本文选题：猪胸膜肺炎放线杆菌 + 细菌分裂　；参考：《湖南农业大学》2015年硕士论文

【摘要】：猪传染性胸膜肺炎是由猪胸膜肺炎放线杆菌感染引起的一种接触性呼吸道疾病,抗生素治疗容易产生耐药性,因此寻找新的药物靶点并设计新的药物非常重要。方法：1、根据已报道的细菌细胞分裂的相关基因的核苷酸序列,通过BLAST了解这些基因在猪胸膜肺炎放线杆菌的分布；根据有关序列设计引物,通过PCR扩增并测序验证这些基因在猪胸膜肺炎放线杆菌是否存在完整的ORF。2、根据GenBank已报道的App ftsZ的有关序列设计引物,从血清3型克隆了ftsZ基因,并进行有关的生物信息学分析。采用DNA重组技术将其重组到载体pET28-(a)表达,提取重组蛋白免疫小鼠制成免疫血清,West blot鉴定细菌是否表达该蛋白。结果：本实验共筛选出胸膜肺炎放线杆菌细胞分裂相关的21种基因,其中大部分分裂相关基因存在于各个血清型中,少数几个基因在不同的血清型有差异。App的ftsZ基因测序、生物信息学分析和细菌提取物的West blot鉴定发现,App的ftsZ基因全长1212bp,含一个开放阅读框(ORF),由起始密码子ATG至终止密子TAA,编码403个氨基酸。编码蛋白的理论分子量为42.6kD,等电点为4.72。与APP的血清1型、4型、5型、8型、10型、11型相似度为96%。系统进化分析发现App不同血清型的ftsZ基因在进化上属于同一分支。蛋白质序列分析发现,FtsZ蛋白在APP各血清型的同源性甚高。结论：本研究为进一步研究胸膜肺炎放线杆菌细胞分裂机制奠定了基础。ftsZ可能成为广谱药物的靶点。
[Abstract]:Porcine infectious pleuropneumonia is a kind of contact respiratory disease caused by Actinobacillus pleuropneumoniae infection. Antibiotic therapy is easy to produce drug resistance, so it is very important to find new drug targets and design new drugs.Methods: according to the nucleotide sequences of reported genes related to bacterial cell division, the distribution of these genes in Actinobacillus pleuropneumoniae was studied by BLAST, and primers were designed according to the relevant sequences.PCR amplification and sequencing were used to verify the existence of complete ORF.2gene in Actinobacillus pleuropneumoniae. According to the sequence of App ftsZ reported by GenBank, primers were designed to clone ftsZ gene from serotype 3, and the related bioinformatics analysis was carried out.The recombinant protein was expressed by DNA recombination technique. The recombinant protein was extracted and immunized to immunize mice to determine whether or not the recombinant protein was expressed by the bacteria.Results: a total of 21 genes related to cell division of Actinobacillus pleuropneumoniae were screened, most of which were found in each serotype, and a few genes were sequenced in different serotypes.Bioinformatics analysis and West blot analysis of bacterial extracts revealed that the ftsZ gene was 1212bp in length, containing an open reading frame (ORF), encoding 403 amino acids from the start codon (ATG) to the terminal codon (TAA).The theoretical molecular weight and isoelectric point of the encoded protein are 42.6 KD and 4.72 respectively.The similarity between APP serotype 1, serotype 1, serotype 4, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 4Phylogenetic analysis showed that the ftsZ gene of different serotypes of App belonged to the same branch in evolution.Protein sequence analysis showed that FtsZ protein had high homology in APP serotypes.Conclusion: this study lays a foundation for further study on the cell division mechanism of Actinobacillus pleuropneumoniae. FtsZ may be the target of broad-spectrum drugs.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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