CAJ | 학술논문

旨在制备高纯度的牛病毒性腹泻病毒（BVDV）NS3蛋白，为建立牛病毒性腹泻 ELISA 检测试剂盒奠定基础，用于区分疫苗免疫和野毒感染。根据 GenBank 中已发表的牛病毒性腹泻病毒非结构蛋白NS3基因设计一对特异性引物，用 PCR 扩增1206 bp 目的片段，克隆到 pET-32a 表达载体，成功构建重组质粒 pET-32a-NS3。将重组质粒转化大肠埃希菌 Transetta（DE3）用 IPTG 诱导表达，通过 SDS-PAGE 分析，结果表明重组 NS3蛋白在体外得到较好表达，表达的融合蛋白分子质量为61 ku。可溶性分析表明重组NS3蛋白以包涵体的形式存在，经镍柱 His 标签蛋白纯化后进行 Western blot 鉴定，结果表明表达的 NS3重组蛋白能与 BVDV 的阳性血清反应。说明所制备的 NS3蛋白反应性好，可用作 ELISA 的包被抗原。
지재제비고순도적우병독성복사병독（BVDV）NS3단백，위건립우병독성복사 ELISA 검측시제합전정기출，용우구분역묘면역화야독감염。근거 GenBank 중이발표적우병독성복사병독비결구단백NS3기인설계일대특이성인물，용 PCR 확증1206 bp 목적편단，극륭도 pET-32a 표체재체，성공구건중조질립 pET-32a-NS3。장중조질립전화대장애희균 Transetta（DE3）용 IPTG 유도표체，통과 SDS-PAGE 분석，결과표명중조 NS3단백재체외득도교호표체，표체적융합단백분자질량위61 ku。가용성분석표명중조NS3단백이포함체적형식존재，경얼주 His 표첨단백순화후진행 Western blot 감정，결과표명표체적 NS3중조단백능여 BVDV 적양성혈청반응。설명소제비적 NS3단백반응성호，가용작 ELISA 적포피항원。
This study aimed to provide pure NS3 protein as antigen for ELISA,which could distinguish BVDV vaccinated from infected cattle.A pair of primers were designed according to the major antigenic re-gion of bovine virus diarrhea NS3 gene derived form GenBank.The NS3 gene with a size of 1 206 bp was amplified by PCR and subcloned in to prokaryotic vector pET-32a.The recombinant plasmid pET-32a-NS3 was transformed in to E.coli Transetta(DE3)and then expressed by IPTG induction.The NS3 recombi-nant protein was analyzed by SDS-PAGE and Western blot.The result showed that the NS3 recombinant protein was mainly expressed in the form of inclusion body and the molecular weight of the fusion protein was about 61 ku.The result of the Western blot showed that the NS3 recombinant protein could be specif-ically recognized by positive serum to BVDV,and may be used as the coating antigen to develop a diagnosis kit against BVDV.