| Porcine circoviruses (PCV) are small nonenveloped DNA viruses containing a unique single-stranded circular genome.Porcine circovirus infection. PCV is the member of the virus family Circovirus of Circoviridae and have2type species:Porcine circovirus type1and2. Type1PCV readily infects but is not known to cause disease in swine; it is the type2that has caused problems in recent years with various disease syndromes which over time results in significant depletion of lymphocytes; post mortem of diseased animals reveals enlarged lymph nodes and abnormal lung tissue. As the epidemiology of PCV2infections is poorly understood, research of PVC2has focused increasingly on the virual detection, investigation of epidemiology and control measures of PCV2infction.To investigate its prevalence in Yangzhou and Taizhou region, Jiangsu province, a PCR method was established by using a pair of primers that derived from the published nucleotide sequence of the PCV2genome, deposited in GenBank. In order to evaluate its specificity, the PCR detection of PCV2was performed using the template DNAs from the reference virus, and the expected products of573bp were only detected in PCV-2, whereas no specific band was detected in PCV1or other virus such as Pseudorabies virus (PRV) and Porcine parvovirus (PPV). Spleen and lymph node samples were obtained from157hoggery in Yangzhou and Taizhou region from July to September2007, and submitted to PCR detection. The data indicated that111of the157herds were in danger of PCV2infection. In addition, PCV2co-infected in inguinal glands and spleen was41.4%, while21.7%just in inguinal glands and only7.6%in spleen. Furthermore,32inguinal glands from the slaughterhouse in Yangzhou were also tested, and the data revealed that the PCV2infection rate in clinical healthy swine was71.88%. All the data sugguested that PCV2were widely infected in the swine, and it appears to work synergistically with parvovirus or other viruses, which perhaps activating a latent form of circovirus or weakening the immune system enough for PCV to take hold.To further investigate the molecular characteristic of PCV2, a pair of specific PCR primers was designed to amplify the complete genome of PCV2. The complete genomes of six PCV2isolates (named TZ60607, TZ60804, YC60711, YZ60615, YZ60721and YZ70717) were amplified and cloned into pGEM-T easy vector respectively. The six recombinant plasmids sequences were analyzed (with the MicroGenic computer program), and compared with the published sequences of PCV2. The data of genomic sequences of the six isolates were shown that their sizes were all1767nt, and all shared96.0%to99.8%identity at nucleoide with the published sequences. In addition, they were more similar to the isolates from China than the strains from the USA and Canada.To obtain the antigen for the demonstration of serum antibody and preparation monoclonal antibodies of PCV, two sets of primers were designed and synthesized according to the capsid protein genes (cap) of porcine circovirus type1and type2(PCV1and PCV2), and two DNA fragment of cap were amplificated from PCV1and PCV2repectively with appropriate restriction sites. The two PCR products were cloned into plasmid vector pGEX-6p-1respectively, with the5’terminus of the cap gene was genetically fused to the3’terminus of the gene coding for the enzyme glutathione S-transferase (GST), which serves as a carrier in this expression system. The resulting plasmids contained the cap gene of PCV1or PCV2were designed as pCAP-PCV1and pCAP-PCV2respectively, and the sequences were analyzed and compared with the published sequences. The data of sequences for the two genes were shown that their size were all in accord with the expected579bp length. In addition, the cap of PCV1was shared100%identity with the sequence of U49186, and the ones of PCV2was in accord with the sequence of DQ104422. The interested recombined plasmids, pCAP-PCV1and pCAP-PCV2, were transferred into E. coli BL21respectively and confirmed that they were all able to express large quantities of a49ku fusion protein (GST-CAP1and GST-CAP2) by SDS-PAGE analysis and Western-blot.To prepare a novel specific mouse anti-PCV monoclonal antibody, female BALB/c mice of8weeks old were immunized with the recombinant capsid protein of PCV2expressed in Escherichia coli BL21. Following the last immunization, the spleen B cells of the mice were fused with Sp2/0myeloma cells. A recombinant capsid protein-based indirect-ELISA was used to screen out the hybridoma cells, and two interested hybridoma cell lines were obtained (designed as M1and G1). The data of Western-blot, dot-ELISA and indirect immunofluorescent assay confirmed that the G1monoclonal antibody can recognized both PCV1and PCV2, while the Ml monoclonal antibody is distinctive specific to PCV2.To detect the serum antibody of swine in clinid, the recombinant capsid protein (His-CAP2) of PCV2which expressed in Escherichia coli BL21(DE3) was used as antigen for developing an indirect-ELISA assay to detect PCV2specific antibody. Conditions of the ELISA were optimized, and suggested that the optimistic concentration of the recombinant capsid protein is6.4μg/mL. Different serum samples were detected for the antibody of PCV2, and it was found that the developed ELISA method is sensitive and specific to the positive serum of PCV2, while not reactive to the negative serum of PCV2and the sera of other virus, such as Classical swine fever virus, Pseudorabies virus, Porcine parvovirus, Porcine reproductive and respiratory syndrome virus. In addition, the data of further tests also confirmed the ELISA method is repeatable within subject and stable between subjects.175serum samples were collected from Suzhong, Jiangsu province, and all submitted to ELISA detection for antibody of PCV2. The data show that123(70.29%) of the175samples were PCV2-specific antibody-positve, which is in according to the data detected by PCR.To research the immunology control of PCV2, virus was cultured in PK15cells and inactivated by formaldehyde. The recombinant capsid protein GST-CAP2were obtained from the Escherichia coli BL21(DE3) which containing the recombinant plasmid pCAP-PCV2.18healthy sixty-day-old pigs (from Jiangquhai hoggery, Jiangsu Animal Husbandry and Veterinary College) were assigned to five groups. The first group was vaccinated with the inactivated PCV2and the second group was immunized with the recombinant protein GST-CAP2, while the third group only injected with normal saline water as control. The sera samples were collected on the5th,10th,15th,20th,25th,30th and35th day after the first immunization, and the antibody specific to the PCV2capsid protein were detected by ELISA by using recombinant capsid protein His-CAP2. The data show that the inactivated PCV2and the recombinant protein GST-CAP2both could induce a better antibody response to PCV2with25days maintaining period. In addition, the effect of different antigens were different in induce animal immune response, while the inactivated PCV2is better than the recombinant protein GST-CAP2. |
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