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家蚕核型多角体病毒BmNPV囊膜蛋白P74膜外区克隆和原核表达 被引量：2

Cloning and Expression of the Outer Membrane Region from Bombyx mori Nucleopolyhedrovirus p74 Gene
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摘要 通过PCR扩增家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)囊膜蛋白p74基因膜外区片段,对切胶纯化得到的DNA目的片段与原核表达载体pET28a进行连接,通过不同浓度的IPTG对含有pET28a-p74重组质粒的大肠杆菌BL21(DE3)进行诱导,对诱导产物进行SDS-PAGE电泳。结果表明p74基因膜外区获得了表达;通过His单抗对原核表达产物进行Western blotting分析,其结果证实诱导蛋白带为融合有组氨酸的目的蛋白。对割胶获得的P74蛋白和免疫佐剂进行充分研磨,以研磨后的匀浆液对昆明小鼠进行皮下多点注射,通过收获的抗血清对BmNPV ODV病毒粒子进行West-ern blotting分析,检测到一条分子量大小为74 kD的特异杂交带,表明获得的多抗可用于P74蛋白功能的进一步研究。 To express the outer membrane region of BmNPV p74 gene,open reading frame 123（ORF123,Bm123）,a specific DNA fragment from BmNPV genome was amplified by PCR.Purified target fragment was cloned into the expression plasmid pET28a,which generated recombinant plasmid pET28a-p74.E.coli BL21（DE3） harboring the plasmid pET28a-p74 was induced with different concentration of IPTG and the induced target protein band was confirmed by analysis of SDS-PAGE and Western blotting.The induced target protein band was excised directly and the antiserum was raised in rats.The total protein from BmNPV ODV was examined by harvested antibodies and a specific protein band about 74 kD was detected,indicating that P74-specific antibody can be used for further function analysis of p74 gene.
作者 李国辉 唐琦 胡朝阳 陈慧卿
机构地区 江苏大学生命科学研究院
出处 《生物技术通报》 CAS CSCD 北大核心 2011年第10期135-138,共4页 Biotechnology Bulletin
基金 校科研启动基金项目(09JDG057)
关键词 家蚕核型多角体病毒 p74基因 P74蛋白 原核表达 抗体制备 Bombyx mori nucleopolyhedrovirus p74 gene P74 protein Prokaryotic expression Polyclonal antibody preparation
分类号 Q78 [生物学—分子生物学]
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参考文献14

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同被引文献30

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生物技术通报
2011年 第10期

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