家蚕核型多角体病毒囊膜蛋白GP64的原核表达

摘要

通过Primer Premier 5.0设计1对特异性引物,用于扩增家蚕核型多角体病毒囊膜蛋白GP64的部分DNA片段,对PCR扩增得到的DNA片段进行纯化,并对纯化后的双酶切DNA片段与经同样双酶切后的原核表达载体pET28a进行连接；对捕获有重组质粒pET28 a-GP64的大肠埃希菌BL21( DE3)细胞进行IPTG诱导,通过SDS-PAGE对导产物进行电泳分析,结果表明扩增的目的片段获得了表达；通过His单抗对诱导产物进行Western Blot印迹分析,其结果表明导蛋白带为融合有组氨酸的目的蛋白.对原核表达的GP64截短蛋白和免疫佐剂进行克分研磨,将充分研磨后的匀浆液时昆明小鼠进行皮下多点注射,以制备的GP64多抗对家蛋核型多角体病毒粒子感染的BmN细胞总蛋白进行Western Blot印迹分析,结果 在杂交膜上出现一条特异杂交带、其分子量大小约为64 ku,表明制备的GP64多抗可用于其动能的进一步研究.%Specific primers were designed to amplify a truncated fragment of silk worm BmNPV GP64 gene and purified with PCR. The purified target DNA fragment and prokaryotic expression vector pET2Sa was subjected to double enzymatic digestion with EcoRl and HindUl and then earned out the ligation; E. Coli BL21{DE3) cells that had caught the recombinant plasmid pET28a-GP64 was induced wilh IPTG and analyzed the induced product with SDS-PACE electrophoiesis. And the results showed that the amplified target fragment was expressed. Western blot analysis using 6 X His monoclonal antibody had confirmed that the induced protein band was the target protein blended with histidine. The cut short prokaryolic expressed protein of GP64 and immune adjuvant were fully ground and the fully ground even plasma was hypodermically injected into Kunming mice at multi-point. The total protein from BmN cells infected wilh BmtiPV was examined by Western Blot analysis using raised GP64 antiserum. A specific hybrid protein band with about 64 ku appeared on the hybrid membrane, indicating that the prepared GP64-specific antibody could be used for further study of its function.