摘要
为克隆和表达兔多杀性巴氏杆菌外膜蛋白ompH2基因,并对其表达产物的反应原性进行研究,用设计的1对特异性引物对兔多杀性巴氏杆菌CVCC500株总DNA进行ompH2基因的PCR扩增。将PCR片段克隆至pMD18-T载体中,进行测序。随后,将该基因克隆到原核表达载体pET-30a(+)中,将重组表达质粒pET30a(+)-ompH2转化至大肠杆菌BL21(DE3)中,用IPTG进行诱导。测序分析证明,得到了1条大小为1 052bp的基因片段,与国内外报道的兔多杀性巴氏杆菌ompH2基因的核苷酸序列相似性达99%以上。重组表达质粒的酶切鉴定、PCR扩增和测序分析表明,重组表达载体构建成功。SDS-PAGE检测结果证实,该基因可以在大肠杆菌中表达,表达产物为分子质量约47ku的融合蛋白。Western-blot分析表明,表达的重组蛋白ompH2具有反应原性。为研究兔多杀性巴氏杆菌外膜蛋白ompH2的免疫学活性奠定了基础。
The aim of this experiment was to clone and express an ompH2 gene of rabbit source Pas- teurella rnultocida ,and to study the reactinogenieity of the expressed product. The ompH2 gene was ampli- fied from the total DNA from the rabbit source P. multocida CVCC500 strain using a pair of specific pri- mers,and the PCR products were cloned into pMD18-T vector and sequenced. The ompH2 gene was then cloned into the expression vector pET-30a(+), transformed into Escherichia coli BL21(DE3), and induced by isopropyl fl-D-thiogalactoside. Sequencing result showed that the amplified ompH2 gene was 1 052 bp in size and shared 99 % homology with the reported ompH2 gene from P. multocida. PCR identification, en- zyme digestion identification and sequencing showed that the recombinant expression vector pET30a(+)- ompH2 was successfully constructed. SDS-PAGE confirmed that the gene was expressed in a form of inclu- sion bodies with about 47 ku in molecular weight. Western-blot analysis showed that the expression protein had reactinogenicity. The results provided the foundation for the research on immunological activity of pro tein ompH2 of the rabbit source P. rnultocida.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2013年第7期728-732,共5页
Chinese Veterinary Science
基金
吉林省科技厅青年基金项目(201201099)
吉林省教育厅项目(吉教科合字[2012]第55号)
吉林省科技发展计划项目(20100230
20111820
20120229)
吉林农业大学青年启动基金项目(201241)
国家现代农业产业技术体系建设专项(CARS-44-E-6)
国家自然科学基金资助项目(31272611)
关键词
兔多杀性巴氏杆菌
ompH2基因
克隆
原核表达
rabbit source Pasteurella multocida
ompH2 gene
cloning prokaryotic expression