9株鸡毒支原体29Ku多肽基因的克隆与序列分析

Cloning and sequence analysis of 29 kilodalton polypeptide gene of Mycoplasma gallisepticam

根据已发表的鸡毒支原体(MG)S6株29Ku多肽基因序列设计了1对引物,以9株(广西分离株5株、标准株4株)DNA为模板进行PCR扩增,均得到802bp的特异性片段,将9株MGPCR产物纯化后克隆到pMD18＿T载体上,得到重组质粒。重组质粒经PCR法和EcorⅠ、SalⅠ双酶切等方法鉴定后,测定了9株29Ku多肽基因序列,并在基因库中S6标准株的29Ku多肽基因序列进行分析比较。结果表明,5株分离株与5株标准株29Ku多肽基因核苷酸序列同源性分别为94 4%～99 9%,推导的氨基酸同源性分别为89 7%～99 2%。从各毒株的进化分析表明,5个分离株与标准强毒株S6、A5969、K1501和PG31强毒株间遗传距离较近,而5个分离株与标准株F疫苗株间遗传距离则较远。

A pair of specific primers were designed according to the sequences of 29?Ku polypeptide gene of MG S6 strains.The specific 802?bp fragment of 29?Ku polypeptide gene products were amplified by the primers from night MG strains.The PCR products were cloned into pMD18_T vetor.The nucleotide acid sequence had been determined after being identified by PCR and restriction endonucleases digestion.The sequence homology was between 94.1?% and 99.9?% in nucleotide acids,between 89.7?% and 99.2?% in amino acids.Phylogenetic tree showed that there are far relationship between F vaccine strain and other night MG virulent strains,but there are close relationship between five Guangxi isolates and four virulent reference strains.