高级检索+

对虾肝胰腺细小病毒衣壳蛋白的原核表达和抗血清制备

Prokaryotic Expression of Capsid Protein of Shrimp Hepatopancreas Parvovirus and Preparation of Antiserum

  • 摘要: 对虾肝胰腺细小病毒(hepatopancreatic parvovirus, HPV)是对虾的重要病原之一。本研究的目的是在大肠杆菌中表达全长的HPV病毒衣壳蛋白(CP),制备针对CP蛋白的多克隆抗体。本研究根据中国对虾(Penaees chinensis) HPV病毒(韩国株)的基因序列,人工合成衣壳蛋白全长cp基因,并亚克隆到pSUMO载体,转化到Rossetta (DE3)菌株诱导表达,获得纯化的SUMO-CP融合蛋白后,经免疫兔获得多克隆抗体,通过SUMO蛋白与琼脂糖介质偶联制备成抗原亲和纯化层析柱,去除非特异性抗体成分,并进一步通过ELISA和Western Blotting对血清效价和特异性进行测定。结果表明成功构建了pSUMO-sHPVCP重组质粒,SUMO-CP融合蛋白在大肠杆菌中是以包涵体形式存在的,WB检测大小正确。Ni柱纯化后蛋白纯度可以达到90%以上,本研究制备了10 mg的SUMO-CP融合蛋白用于多抗制备。经过SUMO蛋白亲和柱去除抗SUMO的交叉反应抗体后,抗CP蛋白抗血清的特异性增强,多抗得率达到5.4 mg以上,抗血清中抗CP蛋白的抗血清滴度为512 000以上,经过WB检测后表明抗体的特异性好。本研究为进一步开展对虾HPV病毒致病机理、免疫制剂及诊断试剂的研究奠定了基础。

     

    Abstract: Hepatopancreas parvovirus(HPV) is one of the important pathogens of shrimp. The purpose of this study was to express full-length capsid protein(CP) of HPV in Escherichia coli and prepare polyclonal antibodies against CP protein. In this study, the full-length capsid protein gene was synthesized based on the sequence of HPV virus(Korean strain) from Penaeus chinensis, which was subcloned into pSUMO vector and transformed into Rossetta(DE3) strain to induce expression. After obtaining the purified SUMO-CP fusion protein, polyclonal antibodies serum were obtained by immunizing rabbits. The antigen affinity purification chromatography column was prepared by coupling SUMO protein with agarose medium to remove non-specific antibody components, and the serum titer and specificity were further determined by ELISA and Western Blotting. The results indicated that the recombinant plasmid of pSUMO-sHPVCP was successfully constructed. The fusion protein of SUMO-CP was proved successfully expressed and existed in the form of inclusion body in E. coli by SDS-PAGE and WB detection. The purity of CP protein was more than 90% after purification with nickel column. Ten milligrams of SUMO-CP fusion protein was obtained for the preparation of polyclonal antibodies. After removal of cross-reactive antibodies against SUMO by SUMO protein affinity chromatography column, the specificity of anti-serum was enhanced. The yield of polyclonal antibodies was more than 5.4 mg. The titer of anti-CP protein in serum was more than 512 000. WB test showed that the specificity against CP of serum was good. This study laid a foundation for further research on the pathog-enesis, immune agents and diagnostic reagents of shrimp HPV.

     

/

返回文章
返回