【摘要】 为深入研究新鉴定的"ORF7"蛋白在猪圆环病毒2型(PCV2)感染中的作用,制备了鼠抗ORF7蛋白的多克隆抗体并进行初步应用。首先,克隆ORF7基因,分别构建了重组原核表达质粒pGEX-6p-1-ORF7和重组真核表达质粒pCDH-CMV-Flag-ORF7。将pGEX-6p-1-ORF7进行诱导表达,确定在IPTG 0.8 mmol/L、37℃下诱导12 h表达量最高,且以包涵体形式表达,分子质量大小约为44 ku。将目的蛋白纯化后免疫Balb/c小鼠,第4次免疫后收集小鼠血清,ELISA方法检测其抗体效价为1∶10000,可用于免疫印迹试验检测pGEX-6p-1-ORF7表达的重组蛋白。应用获得的多克隆抗体通过IFA检测PCV2感染的PK-15细胞中ORF7蛋白的表达,Western blot检测pCDH-CMV-Flag-ORF7和pEGFP-N1-ORF2蛋白的表达,结果表明制备的多克隆抗体可用于IFA和Western blot检测ORF7蛋白。研究结果为探究ORF7在PCV2感染中的作用提供了基础材料。更多还原

【Abstract】 In order to further investigate the role of the newly identified "ORF7" protein in porcine circovirus type 2(PCV2) infection,a polyclonal antibody against mouse ORF7 protein was prepared and applied initially.First,the ORF7 gene was cloned,and the recombinant prokaryotic expression plasmid pGEX-6 p-1-ORF7 and the recombinant eukaryotic expression plasmid pCDH-CMV-Flag-ORF7 were constructed.The expression of pGEX-6 p-1-ORF7 was induced at the IPTG 0.8 mmol/L and 37℃ for 12 h,and expression was in the form of inclusion bodies with a molecular mass about 44 ku.The Balb/c mice were immunized with the target protein after purification,and the sera of the mice were collected after the fourth immunization.The antibody titer was 1∶10 000,which can be used for Western blot to detect the expression of pGEX-6 p-1-ORF7.The polyclonal antibody raised was used to detect the expression of ORF7 protein in PCV2-infected PK-15 cells by IFA.The expressions of pCDH-CMV-Flag-ORF7 and pEGFP-N1-ORF2 proteins were detected by Western blot.The ORF7 protein was detected by IFA and Western blot.This study provided the basis for an in-depth study of the role of ORF7 in PCV2 infection.更多还原