关键词: P1 , ORF2 , ORF3 , RT-PCR , RACE , 免疫组化

Abstract: 【Objective】 The objective of this study is to determine the presence of the open reading frames ORF2 and ORF3 in porcine circovirus type 2-like virus P1.【Method】 After transfection of P1 molecular cloning, extracted and purified the total RNA, then RT-PCR was developed for detection of the RNA with specific primers ORF2 and ORF3, respectively. The products of PCR were recovered using AxyprepTM DNA Gel Extraction Kit, and then Trans5α competetent cells were transformed, the single colony were selected and sequenced after tested positively by PCR. Simultaneously, the 5′- and 3′-ends of P1 ORF2 and ORF3 were amplified using RACE. Furthermore, B-cell epitopes of P1 ORF2 and ORF3 were predicted according to their sequences, and then the corresponding epitope peptides were synthesized using standard stepwise solid-phase synthesis method and coupled to keyhole limpet hemocyanin (KLH) to increase antigenicity. New Zealand White Rabbits were immunized with KLH conjugated peptides to prepare anti-P1 ORF2 and -ORF3 polyclonal antibodies. Conventional indirect ELISA was applied to detect the titer, coated with the synthetic peptide antigen, and then their absorbency was measured under 450 nm wavelength. The highest serum dilution of S/N ≥ 2.1 was identified as the antibody titer. Reactivity of P1 and the two polyclonal antibodies were detected by immunohistochemistry, and the copies of P1 in transfected cells were determined by real-time PCR, simultaneously. 【Result】 The RT-PCR with the specific primers of ORF2 and ORF3, could amplify the 111bp and 128bp target fragments, which had 100% sequence homology with P1 ORF2 and ORF3, respectively. The 5′- and 3′- RACE ends of P1 ORF2 located at sites 11（G） and 168（T), respectively; the 5′- and 3′- RACE ends of P1 ORF3 located at sites 285（T） and 579（A）, respectively. According to the prediction, the amino acid sequences of the synthesized epitope peptides were ORF2: CLSRLPQSERPPGRW and ORF3：CVYPKVRERRVLKMP. Titers of the polyclonal antibodies were both higher than 1:512000, which were prepared from New Zealand White Rabbits immunized with KLH conjugated peptides of ORF2 or ORF3. Besides, the polyclonal antibodies could develop specific chromogenic reaction with P1. Dyeing results of the Blue DAB chromogenic reagent kit were hyacinthine, mostly in cytoplasm and few in nucleus, while the control groups were no chromogenic reaction. The results of real-time PCR showed that P1 could propagate in the transfected cells, and 104 h after transfection harvested the largest proliferation.【Conclusion】 The presence of P1 ORF2 and ORF3 was determined at the transcriptional and protein levels by transcriptional analysis and immunohistochemistry.

Key words: P1 , ORF2 , ORF3 , RT-PCR , RACE , immunohistochemistry