轮状病毒SA11衣壳蛋白VP7的毕赤酵母重组表达及IgY的制备

发布时间：2018-09-17 20:08

【摘要】： 目的真核重组表达轮状病毒(rotavirus,RV)SA11株VP7衣壳蛋白并制备、纯化其卵黄抗体(IgY),并鉴定其活性。方法将SA11标准株在MA104细胞中增殖,收集病毒液。从病毒液中提取RNA,通过逆转录聚合酶链反应(RT-PCR)扩增获得SA11株衣壳蛋白VP7的长度为978 bp的编码基因,将PCR产物连接到pMD18-T载体上,进行测序。将重组体亚克隆到分泌表达载体pPICZαB中,再将重组体转化入大肠杆菌Top10中,用BstXⅠ线性化酶切重组质粒后电转入毕赤酵母X-33中,进行测序。转染成功的菌落再用甲醇诱导表达,亲和层析法纯化重组蛋白抗原,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白免疫印迹(Western blotting)鉴定,用重组蛋白免疫罗曼母鸡,收集鸡蛋,Western blotting鉴定、纯化IgY。体外中和实验鉴定IgY作用。结果细胞增殖液中提取的RNA银染可见11个条带,成功构建了含pPICZαB-SA11 VP7的毕赤酵母X-33,毕赤酵母X-33分泌的融合蛋白被收集、纯化。毕赤酵母X-33表达的SA11 VP7的衣壳蛋白经West blotting验证与预测蛋白相对分子量40 200相符。从鸡蛋中提取的鸡的IgY验证为抗VP7的抗体。纯度可达到95%,1个鸡蛋能产10.2 mg的IgY。在体外实验中证明免疫后的IgY与蚀斑相对减少有关。结论抗重组RV SA11衣壳蛋白VP7和IgY的成功制备为疫苗和诊断试剂的开发奠定了基础。
[Abstract]:Objective to express and prepare VP7 capsid protein of rotavirus (rotavirus,RV) SA11 strain, purify its yolk antibody (IgY), and identify its activity. Methods the standard strain of SA11 was proliferated in MA104 cells and the virus solution was collected. RNA, was extracted from the viral fluid and amplified by reverse transcription-polymerase chain reaction (RT-PCR) to obtain the 978 bp encoding gene of capsid protein VP7 of SA11 strain. The PCR product was ligated to pMD18-T vector and sequenced. The recombinant was subcloned into secretory expression vector pPICZ 伪 B, then transformed into Escherichia coli Top10. The recombinant plasmid was digested with BstX 鈪，

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