丝状支原体丝状亚种SC型脂蛋白Q的N末端定点突变及表达载体的构建  

Site-directed Mutagenesis and Recombinant Expression Plasmid Construction of the N-terminal Gene of the Lipoprotein LppQ of Mycoplasma mycoide subsp.mycoides SC

作　　者：高玉龙[1] 辛九庆[1] 李媛[1] 王砚范[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所国家牛传染性胸膜肺炎参考实验室,哈尔滨150001

出　　处：《畜牧兽医学报》2006年第1期95-99,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家"十五"科技攻关重大专项资助项目(2002BA518A04)

摘　　要：根据已发表的丝状支原体丝状亚种SC型(MmmSC)LppQ基因序列设计引物,从MmmSC HVRI X株中扩增出了LppQ N末端基因,并将其分别克隆到pUC18和pUC19上,利用一步重叠延伸PCR突变方法将其66位的TGA突变为TGG,经克隆与序列测定证实突变成功后,将已突变的LppQ N末端基因插入原核表达载体pET32a的多克隆位点,成功地构建了LppQ N末端基因原核表达载体pET32a-LppQ,为其下一步体外表达奠定了基础。The LppQ N-terminal gene of Mycoplasma mycoide subsp, mycoides SC was amplified from MmmSC HVRI X strain by PCR using the primers designed according to the reported MmmSC LppQ sequence. The products of PCR were cloned into the pUC18 and pUC19 vector respectively. Mutagenesis was performed in a one-step overlap extension PCR. Sequence analysis confirmed the successful mutation from TGA to TGG in amino acid 66 in the LppQ N-terminal gene. The fragments amplified by PCR containing the mutation site were subcloned into the pET32a expression vector. Restriction enzyme, PCR and sequencing analysis indicated that the recombinant expression plasmid pET32a-LppQ was successfully constructed, which may provide a basis of expression of the LppQ N-terminal gene in vitro.

关 键 词：牛传染性胸膜肺炎 丝状支原体丝状亚种SC型 脂蛋白 LppQ 定点突变 

分 类 号：S852.62[农业科学—基础兽医学] Q781[农业科学—兽医学]