胸膜肺炎放线杆菌荚膜多糖输出基因部分序列的克隆鉴定、序列拼接和分析应用  

Cloning,identification,sequence assembly and sequence analysis and application of part gene in capsular polysaccharide export gene of Actinobacillus pleuropneumoniae

作　　者：肖国生[1] 曹三杰[1] 段丽丽[1] 文心田[1] 肖驰[1] 马晓平[1] 杨利[1] 

机构地区：[1]四川农业大学动物科技学院基因芯片实验室动物疫病与人类健康四川省重点实验室

出　　处：《中国兽医学报》2007年第3期325-329,共5页Chinese Journal of Veterinary Science

基　　金：教育部长江学者和创新团队发展计划资助项目(IRT0555-9)

摘　　要：用设计的引物对胸膜肺炎放线杆菌1、3、6、9型标准株的荚膜多糖输出部分基因序列进行了扩增，克隆，测序，分别获得4个型的部分cpxDC基因序列和3型的部分cpxD基因序列。对克隆序列和GenBank中已知序列进行序列拼接，分别拼接出1、3、6型的荚膜输出cpxD基因全序列。通过基因序列分析发现，胸膜肺炎放线杆菌1、3、5、6、9型荚膜多糖输出基因已知序列间的同源性很高，达89％～99．88％。1、3、5、6型cpxD基因的推导氨基酸序列间的同源性为93％～99％。在cpxDC基因的保守区内设计出1对种特异性引物，对胸膜肺炎放线杆菌进行鉴定和检测，结果从8个标准型菌株和3株分离株中均扩出约720bp的阳性片段，其他6种能引起猪呼吸道疾病的细菌均呈阴性。所建立的PCR方法最低检出量为10Pg，最适模板量为5ng（50ptL）。试验结果表明，胸膜肺炎放线杆菌不同血清型间的荚膜多糖输出基因存在着高度的保守性。设计出的种特异性引物能用于胸膜肺炎放线杆菌的检测和鉴定。Different sequences of the capsular polysaccharide export （cpx） gene were amplified and cloned with primers from A. pleuropneurnoniae serotypes 1,3,6 and 9. The complete sequence of the cpxD gene of serotypes I, 3,and 6 was assembled by the bio-software,based on cloned sequences and part sequences in GenBank,respectively. The analysis of sequences showed the homology among serotypes 1,3,5,6 and 9 was from 89% to 99.88%. The homology of the deduced amino acid sequence of assembled cpxD gene among 1,3,5 and 6 was from 93% to 99%. Species-specific primers were designed from the conserved region of known sequences ,and used to detect and iden- tify A. pleuropneurnoniae. Expected 720 bp fragment was amplified from all A. pleuropneurnoniae 8 serotypes refer- ence strains and 3 isolates,but did not from other 6 species bacteria of swine. The sensitivity of the PCR was deter- mined to be 10 pg DNA. The optimal template was 5 ng（50 μL reaction）. The results show that the capsular polysaccharide export gene of different serotypes is highly conserved. The PCR developed in this study is specific and sensitive,and capable of detection and identification of A. pleuropneumoniae.

关 键 词：胸膜肺炎放线杆茵 荚膜多糖输出基因 克隆鉴定 序列拼接 PCR 

分 类 号：Q78[生物学—分子生物学] S852.619[农业科学—基础兽医学]