绵羊肺炎支原体(Mycoplasma ovipneumoniae)丙酮酸脱氢酶E1-α亚单位基因(pdha)的克隆、表达及其免疫学活性测定

摘要

丙酮酸脱氢酶α-亚单位(PDHA)在病原体丙酮酸脱氢酶的催化过程中发挥着重要作用.为表达绵羊肺炎支原体(Mycoplasma ovipneumoniae)PDHA蛋白并测定其免疫学活性,应用PCR方法扩增出绵羊肺炎支原体pdha基因并对其序列进行分析,将pdha基因中色氨酸密码子TGA优化为TGG后进行全基因合成,插入到pET32-a(+)载体上,构建了pET3 2-a(+ )-pdha重组质粒,将重组质粒转化到大肠杆菌(Escherichia coli)BL21中诱导表达PDHA蛋白,并通过免疫印迹及小鼠(Mus musculus)免疫试验对其免疫学活性进行测定.结果pdha基因全长1 125 bp,编码375 aa,(G+C)％为34.76％,第304～306位、379～381位、586～588位、592～594位、625～627位、811～813位、889～891位及964～966位TGA在支原体中编码色氨酸而不是作为终止密码子；基因序列比对及进化树分析显示,绵羊肺炎支原体pdha基因与10种支原体的pdha基因序列同源性为32.6％～85.3％,氨基酸序列同源性为39.3％～90.6％,基因序列和氨基酸序列均与猪肺炎支原体(M.hyopneumoniae)有同源性,分别为85.3％和90.6％；绵羊肺炎支原体pdha基因在33℃、IPTG 0.25 mmol/L诱导6h的表达条件下,表达量最高；重组的PDHA蛋白可与绵羊肺炎支原体高免血清具有免疫印迹条带,在免疫小鼠后血清抗体效价与对照组相比,均显著升高(P＜0.05).本实验首次成功克隆表达了绵羊肺炎支原体pdha基因,并证明其重组PDHA蛋白具有较好的免疫学活性.为绵羊支原体肺炎基因工程疫苗及诊断研究提供候选靶标.%Pyruvate dehydrogenase El-a subunit (PDHA) plays an important role in the catalytic activity of pyruvate dehydrogenase of pathogens. In order to characterize the immunologic activity of the PDHA of Mycoplasma ovipneumoniae, we amplified and sequenced the pdha gene of M. Ovipneumoniae. After optimized with TGG instead of TGA for coding the amino acid of tryptophane, the pdha gene was synthesized and inserted into pET32a (+) vector. The recombinant plasmid pET32-&(+)-pdha was transformed into Escherichia coli BL21 and was then induced to express. The recombinant PDHA was purified and subjected to evaluation of its immunologic activity with immunoblot analysis and immune test in mke(Mus muscxdus). The results showed that the open reading frame (ORF) of pdha gene of M. Ovipneumoniae consisted of 1125 nucleotides, with a G+C content of 34.76%, encoding 375 amino acids. There were 8 TGA codons for tryptophane in the pdha gene (located in 304-306, 379-381, 586-588, 592-594, 625-627, 811-813, 889-891 and 964-966 sites). Comparative analysis of the nucleotide and amino acid sequences of PDHA of M. Ovipneumoniae with those of 10 other Mycoplasma species revealed nucleotide sequence homology from 32.6% to 85.3%, with amino acid sequence homology from 39.3% to 90.6%. M. Ovipneumoniae and M. Hyopneumoniae showed the highest nucleotide and amino acid sequence identity (85.3% and 90.6%, respectively). The recombinant PDHA was expressed in the highest level when the recombinant BL21 was induced by 0.25tnmol/L of IPTG at 33 ℃ for 6 h. The recombinant PDHA protein strongly reacted with the antiserum against M. Ovipneumoniae based on the immunoblot assay, and the antibody titers in the immunized mice were increased significantly (P<0.05) compared with the control. We here cloned and expressed the pdha gene of M.ovipeumoniae for the first time. The results indicate the PDHA possesses strong immunologic activity and may be a candidate antigen for vaccine development.