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Volume 23 Issue 3
June 2008

    Bi-shi FU, Bao-lin LI and Lin-ding WANG. Immunogenicity Analysis of Prokaryotic Expression Products of Kaposi's Sarcoma Associated Herpesvirus orf65[J]. Virologica Sinica, 2008, 23(3): 196-202. doi: 10.1007/s12250-008-2925-z
    Citation: Bi-shi FU, Bao-lin LI, Lin-ding WANG. Immunogenicity Analysis of Prokaryotic Expression Products of Kaposi's Sarcoma Associated Herpesvirus orf65 .VIROLOGICA SINICA, 2008, 23(3) : 196-202.  http://dx.doi.org/10.1007/s12250-008-2925-z
    shu

    卡波氏肉瘤相关疱疹病毒orf65基因原核表达产物的免疫原性分析

    cstr: 32224.14.s12250-008-2925-z
    • 1.

      中国科学院武汉病毒研究所病毒学国家重点实验室，湖北武汉 430071;2中国科学院研究生院，北京 100039

    • 通讯作者： 王林定, wangld@wh.iov.cn
    • 收稿日期： 2007-12-06
      录用日期： 2008-01-06

    • 为了纯化卡波氏肉瘤相关疱疹病毒编码的小核衣壳蛋白并分析其免疫原性，我们在原核表达体系中表达了卡波氏肉瘤相关疱疹病毒编码的羧基端orf65基因产品。含重组质粒pQE-80L-orf65的大肠杆菌经由异丙基硫代-β-D-半乳糖苷诱导表达。融合蛋白通过亲和层析纯化。表达的蛋白和纯化的产品经由SDS-聚丙烯酰胺凝胶电泳鉴定。我们建立了一种ELISA方法来鉴定纯化的orf65的免疫原性。纯化的orf65蛋白用于免疫兔子，并获得抗血清。SDS-聚丙烯酰胺凝胶电泳显示纯化的orf65蛋白约为9 kDa,与预期一致。ELISA分析证明orf65具有免疫原性，orf65的多克隆抗体的免疫原性是对照血清中抗体的4倍以上。这些结果证明纯化的orf65蛋白具有很强的免疫原性，且可被用于筛查普通人群的KSHV感染.

      Abstract

      To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi’s sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA

    Immunogenicity Analysis of Prokaryotic Expression Products of Kaposi's Sarcoma Associated Herpesvirus orf65

    • 1.

      State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China

    • 2.

      Graduate School of the Chinese Academy of Sciences, Beijing 100039, China

    • Corresponding author: Lin-ding WANG, wangld@wh.iov.cn
    • Received Date: 06 December 2007
      Accepted Date: 06 January 2008

      Fund Project: the Knowledge Innovation Program of the Chinese Academy of Sciences 0702121YJ1

    • To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi’s sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA
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      Immunogenicity Analysis of Prokaryotic Expression Products of Kaposi's Sarcoma Associated Herpesvirus orf65

        Corresponding author: Lin-ding WANG, wangld@wh.iov.cn
      • 1. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
      • 2. Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
      • Received Date: 06 December 2007
      • Accepted Date: 06 January 2008
      Fund Project:  the Knowledge Innovation Program of the Chinese Academy of Sciences 0702121YJ1

      Abstract: To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi’s sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA

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