猪肺炎支原体JM株的分离鉴定及主要抗原蛋白基因序列分析

Isolation and identification of Mycoplasma hyopneumoniae JM strain and analysis of its main antigen protein genes

为了对从广东地区若干个屠宰场采集的164份疑似猪肺炎支原体感染的猪肺脏组织进行猪肺炎支原体(Mycoplasma hyopneumoniae,MHp)的分离鉴定,并了解其主要抗原蛋白基因的遗传变异特点,试验先对采集的肺脏组织进行16S rRNA基因PCR检测,阳性样本进行病原的分离培养、形态学观察、双重PCR鉴定、分离株主要抗原蛋白膜蛋白P46全基因与黏附蛋白P97基因R1R2区序列分析及效价的测定。结果表明:164份临床样本经16S rRNA鉴定后102份为MHp阳性,阳性率为62.2%;阳性样本接种江苏二号(KM)液体培养基生长良好,颜色呈规律性变黄,在固体培养基上均能呈现特异性菌落形态;各代次分离菌株经16S rRNA双重PCR鉴定均能获得3个大小约1000 bp的目的条带,测序结果与国内外MHp各参考株核苷酸相似性为96.42%~98.21%,说明3株分离株均属于MHp,且可稳定遗传,分别命名为JM-24、JM-39、JM-44,分离株生长效价可达1×10CCU/mL;3株分离株主要抗原蛋白P46全基因和P97基因R1R2区与各参考株核苷酸相似性分别为96.0%~99.3%、97.8%~99.1%,其中P46全基因与参考株核苷酸同源性最高的为ES-2L株(登录号为CP058578.1),最低的为168株(登录号为CP002274.1)、168-L株(登录号为CP003131.1)、GDZJ01株(登录号为KF032627.1);P97基因R1R2区与参考株核苷酸同源性最高的为F7.2C株(登录号为CP034597.1),同源性最低的为168/GDZJ01株,P97基因R1R2区与国内外各致病菌株演绎的氨基酸序列同源性为72.3%~96.9%,其中分离株JM-44氨基酸序列在128~150位点存在缺失或变异,P46基因与P97基因R1R2区系统发育分析结果均表明JM-44分离株与国内外标准株及疫苗株(J株/168株)亲缘关系较远。说明我国广东地区MHp流行株与目前市场疫苗流行株遗传变异较大,提示现有疫苗对猪支原体肺炎的保护率可能下降。

In order to isolate and identify Mycoplasma hyopneumoniae(MHp)from 164 porcine lung tissues suspected of Mycoplasma hyopneumoniae infection collected from several slaughterhouses in Guangdong Province,and to understand the genetic variation characteristics of its main antigen protein genes,the test first carried out 16 S rRNA gene PCR detection on the collected lung tissues,for the positive samples,isolation and culture,morphological observation,duplex PCR identification,sequence analysis on the whole gene of membrane protein P46 and adhesion protein R1 R2 region of P97 of the major antigen protein of the isolated strain,and titer determination were carried out.The results showed that 102 of 164 clinical samples identified by 16 S rRNA were MHp positive,with a positive rate of 62.2%.The positive samples were inoculated into Jiangsu No.2(KM)liquid medium and grew well;the color turned yellow regularly;and specific colony morphology was shown on the solid medium.After duplex RCR identification of 16 S rRNA,the isolated strains of each generation could obtain three target bands with a size of about 1000 bp.The sequencing results had a nucleotide similarity of 96.42%-98.21%with the reference strains MHp at home and abroad.The results suggested that the three isolates belonged to MHp and could be stably inherited,named JM-24,JM-39,JM-44 respectively.The growth titer of the isolates could reach 1×10CCU/mL.The nucleotide similarity of the P46 whole gene and R1 R2 region of P97 gene of the three isolates with the reference strains were 96.0%-99.3%and 97.8%-99.1%respectively.Among them,the ES-2 L strain(accession number CP058578.1)had the highest nucleotide homology between the whole P46 gene and the reference strains,and the strains with the lowest homology were 168(accession number CP002274.1),168-L(accession number CP003131.1),GDZJ01 strains(accession number KF032627.1);F7.2 C strain(accession number CP034597.1)had the highest nucleotide homology between the R1 R2 region of P97 gene and the reference strains,and the 168/GDZJ01 strain had the lowest homology.The amino acid sequence homology of the R1 R2 region of P97 gene and various pathogenic strains at home and abroad was 72.3%-96.9%,and the amino acid sequence of the isolate JM-44 had deletions or mutations at positions 128-150.The phylogenetic analysis of P46 gene and R1 R2 region of P97 gene showed that the JM-44 isolate was far from the domestic and foreign standard strains and vaccine strains(J strain/168 strain).The results suggested that the genetic variation between the MHp epidemic strains in Guangdong Province and the current vaccine epidemic strains in the market was large,suggesting that the protection rate of the existing vaccine against Mycoplasma hyopneumoniae may decline.