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中文摘要：
以传染性造血器官坏死病毒Sn1203株（IHNV-Sn1203）基因组RNA提取物为模板,利用生物信息学软件分析,通过RT-PCR一步法扩增截短的G蛋白基因序列（约375 bp）,将其克隆到表达载体p ET-27b中,构建重组表达质粒p ET-27b-IHNV-short G,通过大肠杆菌Rosetta表达菌株获得高效表达。在IPTG浓度为0.25 mmol/L时,37℃诱导表达,经SDS-PAGE电泳分析显示目的蛋白相对分子质量约为14 000,符合预期大小,并以包涵体的形式表达,4 h时目的蛋白表达量最大。蛋白经变性、复性处理后获得不带任何标签的纯化蛋白,并利用该蛋白制备兔抗血清。ELISA结果显示,兔抗血清的效价为1∶80 000,说明制备的兔抗血清能够识别表达的重组蛋白;间接免疫荧光结果表明兔抗G蛋白血清具有良好的特异性,并且与VHSV参考毒株没有任何交叉反应。
英文摘要：
The genomic RNA extracted from infectious hematopoietic necrosis virus Sn1203（ IHNV-Sn1203） was used as a template to clone truncated glycoprotein by one-step RT-PCR. The gene which is about 375 bp was cloned into the expression vector p ET-27 b,and the recombinant plasmid p ET-27b-IHNV-short G was highly expressed by E. coli strain Rosetta.The result of SDS-PAGE showed that the protein with expected size 14 k Da was obatained after induced for 4 hours at the IPTG concentration of 0. 25 mmol / L at 37 ℃,and the protein was expressed as the form of inclusion body. The truncated protein was purified by denaturation and renaturation treat using different concentrations of urea,and the protein was obtain without any labels. The purified protein was used to produce antisera. The antisera against glycoprotein prepared in this study could react specifically with both natural glycoprotein of the IHNV-Sn1203 and the recombinant truncated glycoprotein in ELISA test,and the antisera titer against the natural glycoprotein was1∶ 40 000,the recombinant glycoprotein was 1∶ 80000. IFA result showed that the antisera could specifically recognize the glycoprotein on the surface of IHNV-Sn1203 and have no cross-react with VHSV.
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