农业生物技术学报2012，Vol.20Issue(3)：275-282,8.DOI:10.3969/j.issn.1674-7968.2012.03.007
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
Cloning and Expression of Pyruvate Dehydrogenase E 1-α Subunit Gene (pdha) in Mycoplasma ovipneumoniae and Its Immunologic Activity Evaluation
摘要
Abstract
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.关键词
绵羊肺炎支原体/pdha基因克隆/PDHA蛋白表达/免疫学活性Key words
Mycoplasma ovipneumoniae/pdha gene cloning/PDHA expression/Immunologic activity引用本文复制引用
许健,储岳峰,高鹏程,赵萍,贺英,剡根强,逯忠新..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[J].农业生物技术学报,2012,20(3):275-282,8.基金项目
甘肃省科技支撑计划项目(No.1011NKCA054)、甘肃省农业生物技术专项(No.GNSW-2010-09)、国家科技基础性工作专项(No.2008FY210200)和国家现代肉羊产业技术体系(No.CARS-39) （No.1011NKCA054）
