猪圆环病毒II型ORF4真核表达载体构建及其抗体制备

为构建猪圆环病毒II型(PCV2) ORF4基因的真核表达载体，并制备ORF4抗体。用PCR方法扩增PCV2 ORF4基因，将其克隆入真核表达载体pCDNA3.1(+)或pEGFP-C1，成功构建出重组质粒pCDNA-ORF4、pEGFP-ORF4。重组质粒pEGFP-ORF4转染PK15细胞后，pEGFP-ORF4蛋白在细胞中明显表达，荧光信号主要定位于细胞核内。重组质粒pCDNA-ORF4接种BALB/c小鼠后，接种小鼠血清能与PCV2发生特异性免疫反应，经4次接种后抗体效价均达到1:26以上，表明诱导产生了PCV2 ORF4抗体。研究结果表明，构建了2种PCV2 ORF4真核表达载体，并制备了PCV2 ORF4抗体，为PCV2 ORF4基因功能研究奠定了基础。

Construction of Eukaryotic Expression Vector and Production of Antibody to Open Reading Frame 4 of Porcine Circovirus Type 2

The objective of this study was to construct the eukaryotic expression vector of open reading frame 4 (ORF4) of porcine circovirus type 2 (PCV2) and produce the antibody against ORF4. The PCV2 ORF4 gene was amplified by PCR method and cloned into the eukaryotic expression vector pCDNA3.1 (+) or pEGFP-C1, then the recombinant plasmid pCDNA-ORF4 and pEGFP-ORF4 was constructed successfully. After transfection of the recombinant plasmid pEGFP-ORF4 in PK-15 cells, the expression of pEGFP-ORF4 protein was obvious, and the fluorescence signal was mainly localized in the nuclei. After inoculation of the recombinant plasmid pCDNA-ORF4 in BALB/c mice, the specific immune reaction of the inoculated mice sera with PCV2 was observed, and the antibodies titer were all above 1:26 after the fourth inoculation, was indicative of the induction of the antibodies to PCV2 ORF4. The results indicated that two kinds of eukaryotic expression vector of PCV2 ORF4 were constructed and the antibodies against PCV2 ORF4 were prepared, which laid the foundation for researches on PCV2 ORF4 gene functions.