山羊支原体山羊肺炎亚种MCCG-0521乳酸脱氢酶基因的克隆表达及其编码氨基酸的生物学分析  

Prokaryotic Expression of LDH Gene of Mycoplasma capricolum subsp.capripneumoniae MCCG-0521 and Biological Characteristics of Its Protein-encoded Amino Acids

作　　者：刘威[1] 蒋鹏程 杨克礼[1] 袁芳艳[1] 周丹娜[1] 刘泽文[1] 高婷 郭锐[1] 孙裴[2] 田永祥[1] LIU Wei;JIANG Peng-cheng;YANG Ke-li;YUAN Fang-yan;ZHOU Dan-na;LIU Ze-wen;GAO Ting;GUO Rui;SUN Pei;TIAN Yong-xiang(Institute of Animal Science and Veterinary Medicine,Hubei Academy of Agricultural Science/Key Laboratory of Prevention and Control Agents for Animal Bacteriosis(Ministry of Agriculture and Rural Affairs)/Hubei Provincial Key Laboratory of Animal Pathogenic Microbiology,Wuhan,Hubei,430064,China;Anhui Agricultural University College of Animal Science and Technology,Hefei,Anhui,230000,China)

机构地区：[1]湖北省农业科学院畜牧兽医研究所/农业农村部畜禽细菌病防治制剂创制重点实验室/畜禽病原微生物学湖北省重点实验室,湖北武汉430064 [2]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《动物医学进展》2023年第2期35-40,共6页Progress In Veterinary Medicine

基　　金：国家重点研发计划项目(2016YFD0501506);湖北省农业科技创新中心资助项目(2019-620-000-001-17)。

摘　　要：旨在获得山羊支原体山羊肺炎亚种MCCG-0521乳酸脱氢酶(L-lactate dehydrogenase,LDH)的蛋白表达产物,分析其编码氨基酸的生物学特性,为进一步研究LDH蛋白的功能奠定基础。试验完成了LDH基因的克隆和密码子改造,成功将LDH基因中473、761、779 nt处的A突变为G,使改造后的LDH基因能在大肠埃希氏菌Rosetta中正确表达。SDS-PAGE电泳结果显示,IPTG诱导目的蛋白表达后获得一条大小约34 ku的蛋白条带,Western blot结果表明获得的原核表达产物可与抗6×His tag的单克隆抗体发生特异性反应,研究采用DNAStar Protean模块分析了LDH的二级结构、蛋白骨架柔性区域和可能的B细胞抗原优势表位,为进一步研究其功能奠定了基础。This study aims to obtain the protein expression products of Mycoplasma capricolum subsp.capripneumoniae MCCG-0521 L-lactate dehydrogenase(LDH),analyze the biological characteristics of its protein-encoded amino acids,and lay a foundation for further study of LDH functions.Briefly,the cloning and codon modification of LDH gene were completed.Three TGA codons(located in 473 nt,761 nt,779 nt)were mutated into TGG by using site-directed mutagenesis kit,to ensure the correctness of LDH expression in Escherichia coli.The SDS-PAGE results showed that the LDH fusion protein,with a molecular weight of 34 ku,was obtained after IPTG induction.Western blot results showed that the obtained prokaryotic expression product could react specifically with the mouse anti-His tag monoclonal antibody.Furthermore,the secondary structure,backbone flexibility,antigenicity,surface accessibility of the LDH protean were analyzed through DNAStar protean software,and the potential distribution of the dominant epitopes of B-cells was predicted.

关 键 词：山羊支原体山羊肺炎亚种 MCCG-0521 乳酸脱氢酶 密码子改造 二级结构 

分 类 号：S852.62[农业科学—基础兽医学]