The Study Of Porcine Circovirus 2 Type

Postweaning multisystemic wasting syndrome (PMWS) was a novel discovered disease of pigs caused by porcine circovirus 2(PCV2) .It can not only lead to postweaning pigs wasted and died severely, but also was associated with porcine dermatitis and nephropathy syndrome (PDNS) of adult pigs, reproductive failure of pregnant sows, porcine respiratory disease complex, proliferous and necrotic pneumonia and congenital tremor type A2 of piglets. The wide prevalence and spreading of the disease posed a serious threat to pig industry and huge economic loss in many nations and regions in the world. A more serious thing is that pigs infected with PCV2 showed immunodepression and being suffered susceptibly from other virulent pathogens. Till now, PCV2 has become one of the most important pathogens blocking the development of pig industry of our country seriously. In order to determine whether or not the disease has been existed in Shaanxi province and supply pig industry a scientific foundation and technological measurement for diagnosis and prevention of the disease, the study of porcine circovirus 2 has been made and the results was as follows:1. Serological investigation of PCV2 of Shaanxi province. .467 serum samples were collected from pig herds in 7 different areas of Shaanxi province and the antibody titers against PCV2 were detected by indirect ELISA method. The results showed that: the total average positive rate of PCV2 antibody in these 7 different areas of Shaanxi province was 35.9%(171/467); the serum positive rates of Unweaned piglets and Postweaning piglets were 2.3%(1/44) and 48.8% (41/84) respectively; and they were 49.4%(39/79),16.1%(5/31),16.9%(20/118) and 57.5%(69/120)in hogs,service boars,replacement gilts and Sows, respectively. The serum positive rates in different areas were various and ranged from 20% to 65%.2. Isolation of PCV2 Shaanxi isolate. According to the published genomes of porcine circovirus 1 and 2(PCV1,PCV2), two pairs of specific primers were designed to detect PCV2 DNA in tissue samples from several farms by a nested PCR assay. Within 160 samples, 87 samples were PCV2 positive. Three of the positive samples from pigs with typical clinic signs of PMWS were selected and inoculated in the permanent PK-15 cell line culture to pass blindly.PCV2 DNA were detected by PCR from the 10th and 20th passages of inoculated PK-15 cell cultivation. The results indicated that PCV2 Shaanxi strain has been isolated. It was the pathological witness of PMWS incidence in Shaanxi province.3. Cloning and sequencing analysis of ORF2 gene of PCV2 Shaanxi isolate. A pair of primers was designed according to the PCV2 sequences in GenBank. For cloning ORF2 gene into the vector conveniently, Kpn I and Hindâ…¢enzyme digestion sites were added into primer P1 and P2 , respectively. The ORF2 gene of PCV2 Shaanxi isolate was amplified by PCR and the PCR products were cloned into pMD 18-T Easy vector, then identified by PCR amplification, enzyme digestion and sequencing analysis. The recombinant plasmids named pMD-ORF2 were screened. The sequences of the cloned PCR products was analyzed by DNAStar software and compared with other PCV2 isolates in GenBank. The results indicated that: the ORF2 gene sequence of PCV2 Shaanxi isolate was closely related with DB strain(from northeast China), the homology of their nucleotide sequences was 99.7%.Compared with other strains, the homology of nucleotide sequences and deducted amino acids of PCV2 Shaanxi isolate ranged from 90.6% to 99.7% and 90.9% to 99.6% , respectively.4. Establishment of indirect ELISA for detection antibody of PCV2 ORF2 protein. In order to express ORF2 gene of PCV2, a pair of primers was designed according to the sequence of PCV2.The ORF2 gene was amplified by PCR used the primers carrying Hind III and Kpn I enzyme digestion sites and subcloned into prokaryotic expressed vector pBAD/gIII. The recombinant plasmid named pBAD/gâ…¢-ORF2 was constructed. After that, pBAD/gâ…¢-ORF2 was transformed into Escherichia coli TOP10 and expressed being induced by L-Arabinose. The results of SDS-PAGE and Western-blot indicated that the ORF2 gene was expressed and the molecular weight of recombinant fusion protein was about 28 ku. Using the fusion protein, an indirect ELISA method has been built for detecting PCV2 antibody. Used the method, 160 sera from pig farms in Guanzhong and Shaanbei of Shaanxi province were tested and the results were agreed with that detected with commercial ELISA kit produced by Huazhong Agricultural University .5.ORF1,ORF2 gene of PCV2 were amplified by PCR . The PCR products were cloned into pMD18-T vector one by one and the recombinant plasmids were obtained. The interesting gene was subcloned into the shuttle plasmid of pAdTrack-CMV. The recombinant plasmid and adenovirus backbone DNA were cotransformated into E.coli BJ5183 and the recombinant adenovirus genomic DNA was obtained. After transfecting 293 cell line with recombinant adenovirus the genomic DNA by the lipofectam ine method, the recombinant adenovirus carrying ORF1 and ORF2 gene was screened under fluorescent microscopy and PCR analysis. The recombinant adenovirus have been immuned mice, detected sera antibody about PCV2 of the mice by using ELISA. The result show that we obtained ORF1 and ORF2 gene of PCV2 , constructed the pMD18-ORF1-ORF2 and pAdCMV-ORF1-ORF2 vector, and obtained the recombinant adenovirus carrying ORF1 and ORF2 gene.And we find that the recombinant adenovirus may induce the mice acquire specific antibody about PCV2. The research put a solid foundation for development of genetic engineering vaccine of PCV2 infection.6. The study of PCV2 DNA vaccine. According to the amoino acid sequence of nucleolus location signal peptide, and consider the favor code of PCV2 design primer sequence. The MPG-ORF2 gene was amplified by PCR. The interesting gene was cloned into the shuttle plasmid of pAdTrack-CMV. The pcrCMV-MPG-ORF2-SV40end was amplified by two times PCR using pAdTrack-CMV-MGP-ORF2 .The pcrCMV-MPG-ORF2-SV40end was cloned into pMD18-T vector, then obtain the Pmd18-T-CMV-MPG-ORF2-SV40end. Using artificial synthsis MPG and hyaluronic acid enzyme for assist dose to handle the three kinds of DNA vaccines. Then use the vaccines to immunity the rats. The specific antibodies to PCV2 ORF2, the Murine spleen lymphocytes stimulated by concanavalin A (ConA), the NK cells activities and lymphocytes kinds of the rats have been detected. The result showed that desiganed DNA vaccine can induce the rats produce special immunity rection, and the antibody of PCV2 in serm rise obviously. It is the first time ues artificial synthsis peptide and hyaluronic acid enzyme as assist dose for DNA vaccine groped in PCV2 DNA vaccine. And provide useful data for the study of DNA vaccine.