CAJ | 학술논문

猪繁殖与呼吸综合征病毒（PRRSV）TJ 株在细胞克隆纯化后得到非结构蛋白2（Nsp2）缺失的致弱毒株TJM株，根据GenBank数据库中公布的PRRSV TJ株F3代全基因序列设计合成引物，采用 RT-PCR方法扩增了 PRRSV TJM 株缺失片段 dNsp2，利用原核表达载体 pGEX-6p-1构建重组质粒 GST-dNsp2，将重组质粒转入大肠埃希菌 BL21（DE3）中表达，获得了可溶性表达的融合蛋白（GST-dNsp2）。经亲和柱层析纯化后，得到纯化的GST-dNsp2蛋白，含量1．6 mg/mL。用PreScissionTM蛋白酶对融合蛋白GST-dNsp2进行解离，获得浓度为0．41 mg/mL的目的蛋白 dNsp2。对 GST-dNsp2及 dNsp2进行 West-ern blot鉴定，证明表达的蛋白具有良好的反应性。本研究获得的 GST-dNsp2及 dNsp2为 ELISA方法鉴别检测PRRSV TJ株与TJM株提供了基础资料。
저번식여호흡종합정병독（PRRSV）TJ 주재세포극륭순화후득도비결구단백2（Nsp2）결실적치약독주TJM주，근거GenBank수거고중공포적PRRSV TJ주F3대전기인서렬설계합성인물，채용 RT-PCR방법확증료 PRRSV TJM 주결실편단 dNsp2，이용원핵표체재체 pGEX-6p-1구건중조질립 GST-dNsp2，장중조질립전입대장애희균 BL21（DE3）중표체，획득료가용성표체적융합단백（GST-dNsp2）。경친화주층석순화후，득도순화적GST-dNsp2단백，함량1．6 mg/mL。용PreScissionTM단백매대융합단백GST-dNsp2진행해리，획득농도위0．41 mg/mL적목적단백 dNsp2。대 GST-dNsp2급 dNsp2진행 West-ern blot감정，증명표체적단백구유량호적반응성。본연구획득적 GST-dNsp2급 dNsp2위 ELISA방법감별검측PRRSV TJ주여TJM주제공료기출자료。
An attenuated porcine reproductive and respiratory syndrome virus (PRRSV)TJM strain with Nsp2 deletions derived from a highly pathogenic PRRSV TJ strain by cloning and purificationon Marc-145 cells.The specific primers were synthesized according to the sequence of porcine reproductive and respirato-ry syndrome virus TJ strain F3 published in GenBank.The deleted region of PRRSV TJM strain Nsp2 was amplified by RT-PCR and cloned into pGEX-6p-1 to construct the recombinant plasmid GST-dNsp2.The recombinant vector was transformed into E.coli BL21(DE3)and expressed.After purification with affinity column,GST-dNsp2 protein was purified and concentration can be up to 1 .6 mg/mL.The targeted protein dNsp2 was dissociated from fusion protein GST-dNsp2 by the PreScissionTM protease,concentration was 0.41 mg/mL.The results showed GST-dNsp2 and dNsp2 had good immunologic activities.The high level expression of dNsp2 laid a basis for establishment of an ELISA for distinguishing PRRSV TJ strain from TJM strain.