狂犬病病毒糖蛋白富集表位基因克隆及其在原核系统中的表达

Cloning of Enrich Epitope of Rabies Virus Glycoprotein Gene and Its Expression in Prokaryotic Cells

目的构建狂犬病病毒标准强毒株(CVS-24)糖蛋白富集表位基因原核表达系统。方法通过RT-PCR扩增CVS-24株糖蛋白两段表位富集区基因,并将其克隆至载体pET-28a(+),构建重组表达质粒pET-G1和pET-G2,转化大肠杆菌Rosetta,IPTG诱导表达。表达产物分别经12%SDS-PAGE和Western blot检测。结果大肠杆菌可高效表达目的基因,所表达的蛋白可被狂犬病病毒标准阳性血清所识别。经薄层扫描分析,目的蛋白表达量可占菌体总蛋白的42%(G1)和34%(G2)。结论已成功构建了狂犬病病毒标准强毒株(CVS-24)糖蛋白富集表位基因原核表达系统,所表达的目的蛋白具有良好的免疫反应性,有可能作为检测狂犬病病毒血清抗体的候选基因。

Objective To construct a prokaryotic expression system for the enrich epitope of glycoprotein gene of virulent standard rabies virus strain CVS-24. Methods Two gene fragments encoding epitope enrich regions of strain CVS-24 were amplified by RT-PCR and cloned into vector pET-28a（ ＋ ）. The constructed recombinant plasmids pET-G1 and pET-G2 were transformed to E. coli Rosetta for expression under induction of IPTG. The expressed product was identified by 12% SDS-PAGE and Western blot. Results The expressed G1 and G2 proteins contained 42% and 34% of total somatic protein respectively,and were recognized by rabies virus positive serum standard. Conclusion A prokaryotic expression system for the enrich epitope of glycoprotein gene of strain CVS-24 was successfully constructed. The expressed protein showed good immunoreactivity and might be used as a candidate gene for determination of serum antibody against rabies virus.