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.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
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)
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
contagious bovine pleuropneumonia （CBPP）
Mycoplasma mycoides subsp. mycoidesSC （MrnrnSC）
lipoprotein
LppQ
site-dlrected mutagenesis
