鱼类病毒性出血性败血症病毒核蛋白基因的原核表达

Prokaryotic expression of recombinant nucleoprotein of viral hemorrhagic septicemia virus(VHSV)

通过逆转录聚合酶链式反应(RT-PCR)获得编码病毒性出血性败血症病毒(Viral hemorrhagic septicemia virus,VHSV)核蛋白基因,将其克隆至原核表达载体pET28a,并在E.coli BL21(DE3)中得到表达,用SDS-PAGE与Western-blot对表达产物进行鉴定。结果表明,通过RT-PCR扩增获得长度为1 221bp核蛋白基因片段,诱导表达重组质粒pET28a-N,经SDS-PAGE检测,IPTG终浓度为1mmol/L时,诱导5h蛋白表达量最高,获得的目的蛋白大小与N蛋白的预测分子质量一致,约为48ku。诱导后的菌液进行超声波破碎后,将沉淀和上清分别用于SDS-PAGE电泳,结果表明目的蛋白主要以包涵体的形式存在。表达蛋白经过复性、纯化,得到了较高纯度的可溶性蛋白。经Western-blot检测表明,该表达产物能被羊抗VHSV阳性血清特异性识别。

Viral hemorrhagic septicemia virus（VHSV）,the etiologic agent of viral hemorrhagic septicemia in salmonid,caused great loss in aquaculture industry.Nucleoprotein is an important protein for activation of host immunological reaction.The nucleoprotein gene of VHSV was amplified by reverse transcription-polymerase chain reaction（RT-PCR）,and cloned into pET28a vector.The recombinant E.coli BL21（DE3） containing pET28a-N was induced and the expressed protein was detected by SDS-PAGE and Western-blot.The nucleoprotein gene fragment was 1 221 bp in length.The 48 ku target protein was expressed successfully after induced by 1 mmol/L IPTG at 5 hours.But the protein was mainly in the form of inclusion bodies after SDS-PAGE detection.The inclusion bodies was refolded with 8 mol/L urea,and diluted in 0.01 mol/L PBS（pH=9.0）.Western-blot analysis showed that the expressed production can be identified specifically by the goat anti-VHSV positive serum.