Study On Construction Of Porcine Circovirus Infectious Molecular Clone And Inhibition Of Rep Expression By Sirna

Porcine circovirus (PCV) recently emerged as an important infectious pathogen for pigs in the world. It is recognized as the causal agent of post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). PCV also has been associated many other diseases, such as reproductive disorders in sows, porcine respiratory disease complex (PRDC) and congenital tremors (CT). Additional, PCV can damage immune system and induce immunosuppression. Therefore, second infection is very ease to take place in PCV infected pigs. Now, PCV infecton is potentially serious economic impact on the swine industry. It is demonstrated that PCV is very popular in pigs and porcine-derived commercial product. It raises concern for potential human infection through food and xenotransplantation. Unfortunately, there is no efficient method to deal with PCV infection until now. So finding an efficient way to prevent pigs from PCV infection is insistent necessary in the world.In this study, multiplex PCR for detection of PCV2, PRV and PPV, PCV infectious molecule clone and inhibition rep gene expression by siRNA were reported. It will provide usefully information for further research on gene function and immunoprevention of PCV. The main contents of this research are listed as follows.1. Multiplex PCR was used to detection of porcine circovirus type 2, porcine parvovirus and porcine pseudorabies virusMultiplex PCR was established to detect porcine circovirus type 2(PCV2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV) and applied to samples from 137 piglets exhibiting clinical signs of postweaning multisystemic wasting syndrome (PMWS). Results showed that PCV2 DNA was detected from all samples. Moreover, 43 samples were positive for PPV but negative for PRV, 11 samples were positive for PRV but negative for PPV, 35 samples were both positive for PPV and PRV. These results suggest that PCV2 co-infection with PRV, PPV may play an important role in PMWS. Also multiplex PCR is an appropriate candidate method for diagnosis of PCV2, PRV and PPV synchronously in field cases.2. Construction of PCV1 infectious molecule clonePCV1 was isolated from IBRS-2 cells line. Completely genomic sequence was cloned by PCR. Sequence analysis indicated that this PCV1 strain shares >98%nucleotide identity with other PCVl strains in GenBank.Then double copy molecule clone (pSK2PCV1) was constructed and used to transfect PK-15 cell line. The results of indirect immunofluorescence (IIF) and RT-PCR indicated that there was virus replication and gene expression after pSK2PCV1 transfecting PK-15 cell.3. Construction of infectious clone of chimeric porcine circovirus(PCV1-2) Chimeric porcine circovirus molecular clone (pSK2PCV1-2) was constructed bycloning capsid gene of PCV2 into the backbone of PCV1. PK-15 cells was transfected with pSK2PCV1-2 and then cultivated in plate for five passages. mRNA of PCVl rep and PCV2 ORF2 were detected in the fifth passage.but mRNA of PCV1 ORF2 and PCV2 rep were not detected in the cells. On the other hand, capsid protein of PCV2 was also detected by IIF. This study indicated that pSK2PCV1-2 could form infectious virus after transfecting cell.4. Inhibition of rep expression by siRNARep gene of PCV1 was cloned and rep-EGFP fusion was expressed in PK-15cells. It was found that rep-EGFP fusion protein exhibited a diffuse staining pattern throughout the transfected cells. Then three siRNA(A,B,C) were designed to inhibit rep expression in PK-15 cell line. The result of flow cytometry and RT-PCR suggested that rep expression was prominent inhibitted by siRNA, but siRNA(B) and siRNA(C) were more efficient than siRNA(A).5.EGFP as a report gene in PCVl molecular cloneA PCVl molecular clone(pSK2PCVl—EGFP) was constructed by cloning EGFP gene into the C terminal of PCVl ORF2. PK-15 cells was transfected with pSK2PCV1 -EGFP and then cultivated in plate for three passages. Fluorescence was detected in cells nuclear 48 hours after transfection. But no fluorescence can be found in the next passage. It suggested EGFP and capside protein of PCV1 were expressed after tansfection, but it cannot be passed on next passage.