鸡毒支原体S6株P25蛋白粘附特性的研究  

The adhesive character of P25 protein in Mycoplasma gallisepticum S6 strain

作　　者：高利萍[1] 李媛[1] 周长平[1] 刘素丽[1] 邵家日 王琪[3] 陈莹[4] 王显兵[2] 辛九庆[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/动物支原体病创新团队,黑龙江哈尔滨150001 [2]吉林农业大学动物科技学院,吉林长春130118 [3]东北农业大学资源与环境学院,黑龙江哈尔滨150030 [4]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国预防兽医学报》2016年第2期105-110,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：2015兽医生物技术国家重点实验室动物支原体创新团队经费

摘　　要：鸡毒支原体(MG)粘附蛋白是MG定植于宿主细胞表面的关键因子,并在MG致病过程中发挥着重要作用。为筛选并鉴定MG新的粘附蛋白,本研究根据生物信息学软件对MG S6基因组全部序列进行分析,选取了MG的一个假定膜蛋白基因,其大小为675 bp(编码225个氨基酸,分子量约25 ku,将其命名为P25)。通过MG膜蛋白组分的提取及western blot鉴定,结果显示P25分布在MG膜的表面。利用PCR扩增MG p25基因并通过定点突变将其序列中TGA终止密码子突变为TGG(编码色氨酸),通过原核表达其编码的重组P25蛋白(r P25),并以r P25作为免疫原免疫兔子制备高免血清。激光共聚焦显微镜观察显示r P25和MG均对鸡胚成纤维细胞系(DF-1)具有明显的粘附作用,而ELISA和流式细胞仪检测结果则表明r P25和MG对DF-1细胞的粘附为特异性粘附,其中r P25抗血清可以明显抑制r P25和MG对DF-1细胞的粘附作用,从而证明P25为MG的一个粘附相关蛋白。In order to screen and identify new adhesion proteins in Mycoplasma gallisepticum（MG）,a putative membrane protein gene was selected based on the bio-information analysis,whose size was 675 bp（encoding 225 amino acids with a molecular weight about 25 ku,designated P25）.The extraction of MG membrane protein and western blot showed that the distribution of P25 was in the surface of the membrane.In addition,the p25 gene was amplified by SOE-PCR,and a point mutation of TGA（stop codon） to TGG（tryptophan） was introduced into the p25 gene with the designed primers.The prokaryotic expression of p25 gene and the purified recombinant P25（r P25） was used to prepare the anti-serum in rabbit.Laser confocal microscope observed that both r P25 and MG were able to obviously adhere to chick embryo fibroblast（DF-1） cells.However,when r P25 and MG incubated with r P25 anti-serum,the adhesions were specifically inhabited and decreased significantly.TheELISA and flow cytometry tests also confirmed that the adhesion of the protein was a specific adhesion,which proved that P25 is an adhesion related protein of MG.

关 键 词：鸡毒支原体 DF-1细胞 激光共聚焦 ELISA 流式细胞仪 

分 类 号：S852.62[农业科学—基础兽医学]