兔多杀性巴氏杆菌外膜蛋白ompH2基因的克隆与原核表达

Cloning and prokaryotic expression of outer membrane protein gene ompH2 of rabbit source Pasteurella multocida

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.