DNA免疫法制备猪圆环病毒Ⅱ型ORF3抗体

Production of Antibody to Open Reading Frame 3 of Porcine Circovirus Type Ⅱ by DNA Vaccination

为了制备猪圆环病毒Ⅱ型(PCV2)开放阅读框3(ORF3)蛋白的抗体,并为ORF3功能研究奠定基础,用PCR方法扩增PCV2ORF3基因,对其PCR产物用EcoRI与XhoI进行酶切,并对真核表达载体pCDNA3.1(+)进行同样酶切,连接2种目的酶切产物后转化E.coliDH5a感受态,菌落PCR和序列测定结果显示成功构建出重组质粒pCDNA3.1-PCV2ORF3。将该重组质粒接种BALB/c小鼠,用间接免疫荧光(IFA)检测小鼠血清PCV2ORF3抗体的特异性及其效价。IFA结果证实经PCV2ORF3重组质粒接种所制备的小鼠血清是PCV2ORF3的特异性抗体,小鼠血清PCV2ORF3抗体效价在经过4次接种后均达到1:25以上,抗体检测结果表明成功制备了具有较高效价的PCV2ORF3抗体。研究结果提示:可以通过质粒DNA免疫方法来快速、简便地制备PCV2ORF3抗体,为PCV2ORF3抗体制备提供了一种新的有效途径。

The aim of this study was to produce the antibody against the protein of open reading frame 3 （ORF3） of porcine eircovirus type Ⅱ （PCV2）, and lay foundation for research on ORF3 function. The ORF3 gene of PCV2 was amplified by PCR method, and the PCR product was digested by restriction enzyme EcoR I and Xho I. Meanwhile, the eukaryotic expression vector pCDNA3.1 （＋） was digested by EcoR I and Xho I too. Then, the two expected products digested were ligated, and the transformation in the competent cells of E.coli DH5a was conducted. The results of colony PCR and sequence analysis showed that the recombinant expression vector pCDNA3.1-PCV20RF3 was constructed successfully. Thereafter, several BALB/c mice were inoculated with the recombinant plasmid constructed, and the specificity and titer of the antibody to PCV20RF3 were detected by immunofluorescence assay （IFA）. The IFA results demonstrated that the sera collected from the mice inoculated with the recombinant plasmid PCV20RF3 were the antibodies specific to PCV20RF3. Also, the IFA results showed that the antibody titer of PCV20RF3 in the sera of mice were all more than 1：25 after 4 times inoculation, which indicated that the antibodies against PCV20RF3 with relatively high titer was successfully produced. The research results revealed that the antibody to PCV20RF3 could be produced quickly and simply by vaccination of plasmid DNA, and this provided a new effective way to the production of the antibodies to PCV20RF3.