Prokaryotic expression and purification of grass carp reovirus capsid protein VP7 and its vaccine potential
Prokaryotic expression and purification of grass carp reovirus capsid protein VP7 and its vaccine potential
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草鱼呼肠孤病毒衣壳蛋白VP7的原核表达纯化及其疫苗潜力
DOI：
10.5897/ajmr11.221
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发表时间：
2011-07
影响因子：
--
通讯作者：
Lu Liqun
中科院分区：
文献类型：
--
作者：
He Yongxing;Yang Qian;Xu Hongxu;Wu Hao;Wu Fangyuan;Lu Liqun
草鱼呼肠孤病毒（GCRV）的11个dsRNA片段基因组被5个内核蛋白和2个外衣壳蛋白包围。采用谷胱甘肽S-转移酶(GST)融合蛋白表达载体pGEX-4T-3克隆并表达GCRV外衣壳基因vp7，并通过逆转录聚合酶链式反应(RT-PCR)从感染的草鱼中扩增该基因。重组 GST 融合蛋白 rVP7 在 Dh5� 中由 1 mM IPTG 诱导，并使用抗 GST 标签和抗 VP7 单克隆抗血清通过 SDS-PAGE 和蛋白质印迹测定进行证实。预期的52kDa rVP7高表达，并且主要表现在包涵体的形成中。纯化后，将rVP7腹腔注射给实验小鼠，产生抗rVP7多克隆血清。体外微量中和实验表明，rVP7多克隆抗体可以中和GCRV，提示rVP7有潜力作为抗GCRV感染的亚单位疫苗。本研究为进一步表征草鱼草鱼病毒外衣壳蛋白 VP7 的免疫原性铺平了道路，并可基于开发 GCRV 病原体的抗体或抗原检测方法。
The 11 dsRNA fragmental genome of grass carp reovirus (GCRV) is enclosed in five inner core proteins and two outer capsid proteins. The Glutathione S-transferase (GST) fusion protein expression vector pGEX-4T-3 was employed to clone and expression of GCRV outer capsid gene vp7, which was amplified by reverse transcription-polymerase chain reaction (RT - PCR) from infected Grass carp. The recombinant GST-fusion protein rVP7 was induced by 1 mM IPTG in Dh5� and confirmed by SDS-PAGE and Western blot assays using both anti-GST-tag and anti-VP7 monoclonal antisera. An expected 52kDa rVP7 was highly expressed, and was mainly exhibited in the formation of the inclusion body. After purification, rVP7 was intraperitoneally injected to the experimental mice to produce anti-rVP7 polyclonal serum. In vitro microneutralization assay indicated that polyclonal antibody against rVP7 could neutralize GCRV, and suggested that rVP7 had the potential to be used as subunit vaccine against GCRV infection. The present study paved the way for further characterization of the immunogenicity of viral outer capsid protein VP7 in grass carp Ctenopharyngodon idellus and could be based to develop antibody or antigen detection assays for GCRV pathogen.