猪肺炎支原体表面蛋白P46基因的克隆与序列比较

Cloning and sequencing of surface protein P46 gene of Mycoplasma hyopneumoniae

猪肺炎支原体 (MycoplasmaHyopneumoniae ,Mhp)兔化弱毒株R6 5 9株、济南强毒株、强毒Z株、国际标准株2 32 ,通过A2 6培养基培养 ,提取DNA ;利用PCR技术从四株猪肺炎支原体中均能扩增出目的条带。将该序列克隆到pGEM_T_Easy载体上测序。结果表明 ,克隆序列全长 115 2bp ,编码 383个氨基酸和一个终止子 (TAA) ;该序列中含有 3个TGA编码Trp ,而不是终止密码子。比较兔化弱毒株与济南强毒株、国际标准株 2 32及NCBI上发布的J株的P4 6基因序列 ,发现它们的同源性分别为 98 6 %、99 2 %、99 2 % ;比较它们编码的氨基酸发现它们的同源性分别为 98 7%、99 2 %、99 2 %。结果表明猪肺炎支原体的P4 6基因在猪肺炎支原体种内是很保守的 ,因此建立以P4 6蛋白为诊断抗原的ELISA具有潜在的意义。

Mycoplasma hyopneumoniae(Mhp)lapinized strain,virulent JiNan strain,virulent Z strain,standard 232 strain were cultured in A26 liquid medium and DNA was extracted.By PCR technique,the bands of interest were amplified from four strains of Mycoplasma hyopneumoniae.The sequence of interest was cloned into pGEM-T-Easy vector and sequenced.The results of sequencing demonstrated that the cloned sequence is 1 152 bp,coding 383 amno acids and one terminor TAA,and including three TGA codons coding Trp but terminor.Comparing the P46 sequences of lapinized strain with those of JiNan strain,standard 232 strain and J strain whose sequence was publicated in the NCBI web,the result showed the gene sequence identity of lapinized strain with JiNan strain 232 strain and J strain was 98.6 %,99.2 %,99.2 % respectively;comparing the amino acid sequence coded by the sequence showed the identity of them was 98.7 %,99.2 %,99.2 %,respectively.All the results showed Mhp P46 gene is highly conserved in Mhp specie.So the establishment of ELISA based on P46 protein as diagnotic antigen will be of significance potentially.