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[目的]构建传染性脾肾坏死病毒(ISKNV)ORF086基因的融合表达载体,制备抗体为进一步研究ORF086蛋白的免疫保护性提供前提条件。[方法]PCR扩增目的基因,构建重组质粒,经酶切鉴定,PCR和核酸序列分析后,IPTG诱导表达,Ni-柱纯化,注射新西兰白兔获得抗血清。[结果]扩增出ORF086基因,成功构建了重组质粒pET32a-ORF086,SDS-PAGE电泳和Western-blot分析显示,获得一条36.0 kD的融合蛋白。[结论]成功构建了原核表达载体,融合蛋白主要以包涵体的形式存在,经柱层析纯化蛋白,纯度达90%以上。
[Objective] To construct the fusion expression vector of ORF086 gene of infectious spleen and kidney necrosis virus (ISKNV) and prepare the antibody to provide the precondition for further study on the protective immunity of ORF086 protein. [Method] The target gene was amplified by PCR, and the recombinant plasmid was constructed. After restriction enzyme digestion, PCR and nucleic acid sequence analysis, the recombinant plasmid was induced by IPTG and purified by Ni-column. [Result] The ORF086 gene was amplified and the recombinant plasmid pET32a-ORF086 was successfully constructed. A 36.0 kD fusion protein was obtained by SDS-PAGE electrophoresis and Western-blot analysis. [Conclusion] The prokaryotic expression vector was successfully constructed. The fusion protein mainly existed in the form of inclusion body. The purity of the protein was over 90% by column chromatography.