关键词: 猪圆环病毒2型; ORF2; 重组腺病毒

Abstract: In order to research the feasibility of recombinant adenovirus containing Cap gene of porcine circovirus type 2 as genetically engineering vaccine, we designed a pair of primers according to PCV2 sequence. The primers were used for the amplification of the porcine circovirus type 2 ORF2 gene. The target gene was inserted into vector pMD18-T and then subcloned to the adenovirus shuttle plasmid pShuttle-CMV to construct the recombinant shuttle plasmid pShuttle-CMV-ORF2. After the vector was confirmed correct by PCR, restriction endonuclease digestion and sequencing, lineared by PmeⅠenzyme digestion and transformed into E.coli BJ5183-AD-1 containing plasmid pAdeasy-1 to form the homogeneous recombinant adenovirus plasmid vector pAd-CMV-ORF2. It was confirmed correct by PCR, PacⅠ digestion and sequencing. The PacⅠ digestion of linearized pAd-CMV-ORF2 was transfected into AD293 cells using lipofectamine ^TM2000 for virus packaging and amplification. The target gene and its expression in virus were detected by PCR, RT-PCR, IFA and Western blotting. Three times plaque experiments purified the recombinant adenovirus. The TCID50 was 10-8.75/0.1 mL. It was indicated that the recombinant virus carrying the gene encoding the cap protein was successively constructed, and inoculated SPF-class female BALB/c mice with the recombinant adenovirus, and then detected specific antibody level with the ELISA antibody test kit. In these ways, immunization experiments in mice had measured the high levels of specific antibodies, thus provided basis for the further study with genetic engineering of recombinant adenovirus for the PCV2 vaccine.

Key words: porcine circovirus type 2; ORF2; recombinant adenovirus