猪肺炎支原体P46基因的突变及序列分析

Mutation and Sequence Analysis of Mycoplasma Hyopneumoniae P46 Gene

目的:将猪肺炎支原体P46基因中编码Trp的密码子TGA突变为TGG,为P46蛋白的研究及猪肺炎支原体抗体检测方法的建立奠定基础。方法:参考GenBank登录的猪肺炎支原体(Mycoplasma hyopneumoniae Mhp)P46基因序列,利用Primer5.0软件设计合成一对引物,对猪肺炎支原体强毒株F60P46基因的编码区进行扩增,后又设计了三对突变引物,通过重叠延伸PCR(SOE-PCR)对猪肺炎支原体P46基因的三个位点进行定点突变。结果:得到的序列与GenBank中登录的P46基因的核苷酸及氨基酸序列进行序列比较,结果表明它们有较高的同源性。突变后测序结果表明已成功将猪肺炎支原体P46基因中编码Trp的密码子TGA突变为TGG。

Objective： Mutated TGA which encoded Trp to TGG ,lay the foundation for the study of the P46 protein and the method of detecting the Mhp antibody. Methods： Based on the gene sequence of Mycoplasma hyopneumoniae （Mhp） P46 reported in GenBank, primers were designed using Primer 5.0, then the gene of F60 P46 was amplified, Then three mutagenesis primers were designed, the site-directed mutagenesis was performed by overlap-extension PCR （SOE-PCR）method at three sites of Mycoplasma hyopneumoniae （Mhp）P46. Results： Sequence analysis was performed between the F60 P46 gene and the P46 genes reported in GenBank, the results showed that they had high homologous .after mutating, Sequencing results showed that TGA which encoded Trp were mutated to TGG. Conclusion： Myeoplasma hyopneumoniae（Mhp） P46 gene were successfully mutated and Sequencing results showed that they had high homologous with other genes reported in GenBank.