CAJ | 학술논문

目的表达口蹄疫病毒3D聚合酶,并制备兔抗3D多克隆抗体.方法采用PCR方法扩增口蹄疫病毒(FMDV)3D聚合酶基因,经BamHⅠ和NdeⅠ双酶切后插入pET-30a(+)原核表达载体,构建的pET-3D重组表达质粒转化大肠杆菌BL21(DE3)菌株,经IPTG诱导表达,SDS-PAGE鉴定目的蛋白的表达并提取包涵体,纯化目的蛋白.然后用纯化蛋白免疫新西兰大耳白兔制备多克隆抗体,以Western blot鉴定抗体特异性,并以间接ELISA方法测定其效价.结果 SDS-PAGE电泳分析显示,表达的目的蛋白约为46ku,与预期结果相符,制备的多克隆抗体效价达1:8000以上,且具有较高的特异性.结论制备了具有高亲和性和特异性的3D聚合酶多克隆抗体,为3D聚合酶的生物学功能和抗原表位研究奠定了基础.
목적 표체구제역병독3D취합매,병제비토항3D다극륭항체.방법 채용PCR방법확증구제역병독(FMDV)3D취합매기인,경BamHⅠ화NdeⅠ쌍매절후삽입pET-30a(+)원핵표체재체,구건적pET-3D중조표체질립전화대장간균BL21(DE3)균주,경IPTG유도표체,SDS-PAGE감정목적단백적표체병제취포함체,순화목적단백.연후용순화단백면역신서란대이백토제비다극륭항체,이Western blot감정항체특이성,병이간접ELISA방법측정기효개.결과 SDS-PAGE전영분석현시,표체적목적단백약위46ku,여예기결과상부,제비적다극륭항체효개체1:8000이상,차구유교고적특이성.결론 제비료구유고친화성화특이성적3D취합매다극륭항체,위3D취합매적생물학공능화항원표위연구전정료기출.
To prepare the polyclonal antibody against the 3D polymerase of foot and mouth disease virus (FMDV), the 3D polymerase gene of this virus was amplified by PCR and doubly digested with BamH I and Nde I. Then, it were cloned into expression vector pET-30a(+) to obtain the recombinant plasmid pET-3D and this plasmid was transformed to E.coli BL21(DE3) with induction by IPTG.The target protein was identified and purified with SDS+PAGE, and the inclusion bodies were extracted. The purified target protein was used as antigen to immunize New Zealand rabbits to prepare the polyclonal antibody against 3D polymerase of FMDV, which was then characterized by indirect ELISA and Western blotting. As demonstrated by SDS-PAGE, the target protein with a molecular weight at 46 ku was expressed. The polyclonal antibody showed high affinity and obvious specificity and its titer was above 1:8 000. This polyclonal antibody may lay a foundation for the further studies on the biological functions and epitopes of the 3D polymerase of FMDV.