猪肺炎支原体中国分离株热休克蛋白Hsp70(Dnak)的全基因克隆及C末端基因的表达与免疫活性分析  

Cloning and partial expression of the chaperones protein DnaK a Chinese strain of Mycoplasma hyopneumoniae

作　　者：李媛[1] 张建华[1] 王亮[1] 乔祖建[1] 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/动物细菌病研究室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2008年第3期169-173,共5页Chinese Journal of Preventive Veterinary Medicine

摘　　要：猪肺炎支原体(Mycoplasma hyopneumoniae,MHP)是猪气喘病的病原。DnaK又称为热休克蛋白Hsp70,具有分子伴侣和免疫的作用。以MHP中国分离株Yin-1为模板,扩增了DnaK基因的全序列,将该序列克隆到pMD18-T载体上并测序。测序结果Yin-1株DnaK基因即Hsp70基因全长1 803 bp,编码600 aa,第631～633位TGA在支原体编码色氨酸而不是作为终止密码子。将该序列与多种致病支原体DnaK基因进行分析比较,发现Yin-1株与232株、J株、7 448株等MHP菌株的DNA同源性为99.3%～99.4%,氨基酸同源性为99.2%～99.3%,而与非同种支原体的DNA和氨基酸同源性仅为64.5%～74.4%和59.3%～77%。这表明DnaK基因在种内高度保守,种间差异较大。以pET28a为载体构建重组质粒,原核表达DnaK C末端大小为1 kb左右的基因,对表达的蛋白进行纯化后,通过Western blot检测它的免疫活性。原核表达得到大小为41 ku的目的蛋白,经Western blot证实,纯化蛋白可与MHP阳性猪血清反应,具有免疫活性。Mycoplasma hyopneumoniae is the primary cause of Mycoplasma pneumoniae of swine （MPS）. To facilitate studies on the Chaperones protein DnaK （heat shock protein70）, the complete DnaK gene was PCR amplified from the Chinese Yin-1 strain of Mycoplasma hyopneumoniae and cloned into pMD18-T vector and sequenced, The DnaK gene was 1 803 bp in length that encodes 600 amino acids, including TGA codon coding a Trp at the terminus. The gene was highly conserved within Mhp and shared 98.9 %-99.1% nucleotide and 99.2 %-99.3 % amino acid sequence identities with the DnaK genes of the 232, J and 7448 strains, respectively. The 1 kb C terminal fragment was subcloned into expression vector pET-28a, The recombinant protein had Mw of a 41 ku and could react positively with MPS antiserum in Western blot

关 键 词：猪肺炎支原体 猪喘气病 伴侣蛋白DnaK 热休克蛋白70 

分 类 号：S852.67[农业科学—基础兽医学]