| 摘要: |
| 【目的】对小反刍兽疫病毒（Peste des petits ruminants virus，PPRV）H蛋白胞外区（tH）细胞膜受体进行鉴定，为PPRV致病机制的研究奠定基础。【方法】克隆PPRV H基因胞外区（tH），在毕赤酵母(Pichia pastoris)中进行真核表达，纯化后免疫家兔，获得兔抗PPRV tH蛋白特异性抗体；提取山羊外周血淋巴细胞膜蛋白，经SDS-PAGE检测后，湿转印法转印至NC膜，分别利用纯化的重组tH蛋白和PPRV进行病毒铺覆蛋白结合试验（VOPBA），对受体进行鉴定。【结果】成功克隆了1 653 bp的PPRV tH基因，构建其重组酵母表达质粒pPIC9K-tH，诱导表达后获得了60 ku目的蛋白。重组tH蛋白免疫家兔后获得了效价为1∶200的抗血清。Western blotting分析发现，该重组蛋白可与PPRV多克隆抗体发生特异性反应，山羊外周血淋巴细胞膜蛋白上有2个与PPRV和重组tH蛋白结合的蛋白带，分子质量约为38和100 ku。【结论】从山羊外周血淋巴细胞膜蛋白上鉴定到了2种与PPRV结合的蛋白组分，其特性有待于进一步研究。 |
| 关键词: 小反刍兽疫病毒 病毒受体 病毒铺覆蛋白结合试验(VOPBA) |
| DOI： |
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| 基金项目:农业公益性行业科研专项(201103008)；现代农业产业技术体系建设专项(CARS-40-10) |
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| Identifitation of peste des petits ruminants virus receptor from cells membrane |
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MENG Xuelian, DOU Yongxi, ZHAI Junjun,et al
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Chinese Academy of Agricultural Sciences
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| Abstract: |
| 【Objective】The research identified the receptor on the cell membrane that interacts with an extracellular region of H protein of peste des petits ruminants virus(PPRV).It will lay a foundation for studying the pathogenesis mechanism of PPRV.【Method】The extracellular fragment of H gene(tH gene)of PPRV Nigeria75/1strain was cloned and expressed in yeast GS115.Polyclonal-antibody to PPRV was prepared with immunizing rabbits by injecting the purified tH protein.After SDS-polyacrylamid gel electrophoresis(SDS-PAGE),the extracted peripheral blood lymphocyte(PBL)cell membrane proteins were transferred to nitrocellulose membrane;and a virus overlay protein binding assay(VOPBA)was performed by the recombinant tH protein and PPRV to identify the receptor protein in PBL cell membrane.【Result】The size of the extracellular fragment of H gene(tH gene)was 1 653 bp.The recombined yeast expression plasmid pPIC9K-tH was constructed successfully.After the recombined strains were induced by 1% methanol,a molecular weight of 60 ku in medium supernatant could be specifically recognized by polyclonic antibody against PPRV.The anti serum with a titer of 1∶200 was obtained by the recombinant tH protein immunized rabbits.The PBL cell membrane proteins were bound with the purified tH protein and PPRV,and two clear protein bands of 38 ku and 100 ku were found.【Conclusion】There were two proteins binding to PPRV from PBL cell membrane.The characteristics of these two proteins remain to be studied further. |
| Key words: peste des petits ruminants virus(PPRV) virus receptor virus overlay protein binding assay(VOPBA) |
