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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (20): 77-82.doi: 10.11924/j.issn.1000-6850.2012-1385

所属专题： 生物技术； 畜牧兽医

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

猪肺炎支原体地方毒株P97-R1基因序列分析及原核表达

贾丽艳   

  • 收稿日期:2012-04-11 修回日期:2012-04-17 出版日期:2012-07-15 发布日期:2012-07-15
  • 基金资助:

    山西省教委科技项目

Sequence Analysis and Prokaryotic Expression on P97-R1 gene of Mycoplasma hyopneumoniae

  • Received:2012-04-11 Revised:2012-04-17 Online:2012-07-15 Published:2012-07-15

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摘要：

为克隆和表达猪肺炎支原体(Myeoplasma hyopneumoniae) 地方毒株Z株P97-R1基因，探索不同菌株P97-R1基因的差异及其蛋白作为诊断抗原的可能性，本研究根据GenBank登陆的猪肺炎支原体(Mhp)232株P97基因，使用Primer Permier 5.0和DNAman设计引物，以猪肺炎支原体地方毒株Z株基因组DNA为模板，用PCR方法扩增出了P97-R1基因片段，并将该基因片段连接到T-easy和pET-32a载体上分别进行测序和原核表达。测序结果与参考株232株序列对比分析发现Z株P97-R1区发生了多处碱基突变；经DNAstar软件分析表明Z株包含11个5aa重复序列，232株P97-R1区包含15个5aa重复序列，推测Rl重复序列的差异与菌株毒力强弱有关。Z株P97-R1基因原核表达产物经SDS-PAGE分析，得到了分子量约为30 KD的蛋白，经Western-blot鉴定表明该表达蛋白具有免疫原性。这为Mhp的诊断、预防及进一步深入研究奠定了基础。

关键词: 综合评价, 综合评价

Abstract:

To express P97-R1 gene on Z strains of Mycoplasma hyopneumoniae for the exploration of possibility of use as a diagnostic antigen, a pair of primers were designed according to the online database of P97 genes of Mhp232 on GenBank, a strain of Mycoplasma hyopneumoniae, and subsequently contributed to the successful amplification of interest segments using the genomic DNA of Z strains of Mhp as templates by PCR. P97-R1 gene was ligated with T-easy, sequenced and was used to transform competent E. coli Rosetta .The results was compared with 232 referential strains. It was revealed that there were many sites of mutations in the P97-R1. There were 11 5-aa repeats in that of the Z strain ,15 5-aa repeats in the P97-R1 region of the 232 strains as analyzed by DNAstar software. Speculated that differences in repeat sequence R1 may be the reasons of the strain was strong or weak. Synthesized products were analyzed using SDS-PAGE and 30 KD proteins of the P97-R1 was detected. The reactionogenicity was then verified by Western-blotting. This work laid a foundation for the diagnosis, prevention Mhp and further research the Mycoplasma hyopneumoniae.

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