CAJ | 학술논문

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).方法将SA11标准株在MAl04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.将重组体哑克隆到分泌表达载体pPICZαB中,再将重组体转化人大肠杆菌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IsY.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Western blotting验证与预测蛋白相对分子质量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.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.
목적 진핵중조표체륜상병독(rotavirus,RV)SA11주VP7의각단백병제비、순화기란황항체(IgY).방법 장SA11표준주재MAl04세포중증식,수집병독액.종병독액중제취RNA,통과역전록취합매련반응(RT-PCR)확증획득SA11주의각단백VP7적장도위978 bp적편마기인,장PCR산물련접도pMD18-T재체상,진행측서.장중조체아극륭도분비표체재체pPICZαB중,재장중조체전화인대장간균Top10중,용BstXI선성화매절중조질립후전전입필적효모X-33중,진행측서.전염성공적균락재용갑순유도표체,친화층석법순화중조단백항원,십이완기류산납취병희선알응효전영(SDS-PAGE)화단백면역인적(Western blotting)감정,용중조단백면역라만모계,수집계단,Western blotting감정、순화IsY.결과 세포증식액중제취적RNA은염가견11개조대,성공구건료함pPICZαB-SA11 VP7적필적효모X-33,필적효모X-33분비적융합단백피수집、순화.필적효모X-33표체적SA11 VP7적의각단백경Western blotting험증여예측단백상대분자질량40 200상부.종계단중제취적계적IgY험증위항VP7적항체.순도가체도95%,1개계단능산10.2 mg적IgY.결론 항중조RV SA11의각단백VP7화IgY적성공제비위역묘화진단시제적개발전정료기출.
Objective To prepare eukaryotic expression of rotavirns (RV) SA11 capsid protein VP7 ,and to generate and purify yolk immunoglobulin (IgY) antibodies against the recombinant VP7 from Roman hens. Methods MA104 cells were infected with the standard SA11 strain and the culture fluid was collected. A DNA fragment of 978 bp encoding SA11 VP7 was obtained by RT-PCR amplification from genomie RNA of RV SA11. The PCR products were iigated to pMD18-T vector following the confirmation by DNA sequencing and sub-cloned into pPICZαB. The recombinant pPICZαB-SA11 VP7 was transformed into E coli Top10. The plasmids were linearized by digestion of BstXI and transformed into Pichia paaoris X-33 through electroporation by DNA sequencing. The transformants were induced with methanol for expression. The cultural supernatant was subjected to SDS-PAGE and Western blotting. Fusion expression was purified through the column of affinity chromatography. IgY was identified and purified by SDS-PAGE and Western blotting from eggs of Roman hens immunized with recombinant SA11 VP7. Results The RNA extracted from the RV culture fluid consisted of 11 bands visualized by silver staining. The expression vector pPICZαB-SA11 VP7 was constructed and the fusion protein in Pichia pastoris X-33 was harvested and purified. The recombinant SA11 VP7 with molecular weight of 40 200 was identified by Western hlotting. The IgY antibodies against the recombinant SA11 VP7 were produced with a purity of 95 percent and yield of 10. 2 mg per egg. Conclusion The preparation of lgY antibodies to recombinant SA11 VP7 might lay a foundation for the development of vaccines and diagnostic techniques.