摘要
目的克隆牛布氏菌virB8基因并在大肠杆菌中进行表达。方法从牛布氏菌S19基因组DNA中PCR扩增virB8基因片段,插入pEASY-E1载体中,构建重组表达质粒pEASY-virB8,转化E.coli BL21(DE3),IPTG诱导表达。表达产物经HisTrapTMFF纯化后,进行SDS-PAGE分析和Western blot鉴定。结果扩增的virB8基因大小为720 bp;重组表达质粒pEASY-virB8经双酶切及测序证实构建正确;表达的重组蛋白相对分子质量约为30 000,表达量占菌体总蛋白的17.3%;纯化的重组蛋白纯度为85%,Lowry法测定蛋白浓度为1.52 mg/ml,可与鼠抗His单抗特异性结合。结论成功克隆了牛布氏菌virB8基因,并在大肠杆菌中表达了重组蛋白,为新型疫苗的研发及布氏菌鉴别诊断方法的建立提供了有效的候选抗原。
Objective To clone the virB8 gene of BruceUa abortus and express in E. coli . Methods The virB8 gene was amplified by PCR from genomic DNA of B. abortus S19 and inserted into pEASY-E1 vector. The constructed recombinant plasmid pEASY-virB8 was transformed to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed product was purified by HisTrapTM FF chromatography and identified by SDS-PAGE and Western blot. Resultus The length of amplified virB8 gene was 720 bp. Restriction analysis and sequencing proved that recombinant plasmid pEASY-virB8 was constructed correctly. The expressed recombinant protein, with a relative molecular mass of about 30 000, contained 17. 3% of total somatic protein and reached a purity of 85% after purification. At a protein concentration of 1. 52 mg/ml determined by Lowry method, the expressed product showed specific binding to mouse monoclonal antibody against His. Conclusion The virB8 gene of B. abortus was successfully cloned and expressed in E. coli, which provided an effective candidate antigen for development of novel vaccines.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第7期841-843,847,共4页
Chinese Journal of Biologicals
