Construction Of PCV1-2 Chimeric Virus Infectious Clone Plasmid With Genetic Markers

Porcine circovirus type 2(PCV2)belongs to Circoviridae Circovirus,a single-stranded negative-chain DNA virus.PCV2 can cause sow reproductive disorders,weaning piglet multi-system failure syndrome,porcine dermatitis nephrotic syndrome,porcine respiratory syndrome and PCV2-related enteritis and other diseases,to the world's pig industry caused huge economic losses.At present,the vaccine is still the main means of prevention and control of PCV2,China's vaccine is mainly inactivated vaccine,the vaccine on PCV2 prevention and control played an important role.However,the current inactivated vaccines also have defects such as inability to effectively induce cellular immunity,and the resulting antibodies can not be distinguished from wild viruses.As a new genetically engineered vaccine,i DNA vaccine can play the role of nucleic acid vaccine and play the role of attenuated vaccine,which can induce humoral immunity and cellular immunity at the same time,and has good prospects for development.In this study,a PCV1-2 chimeric viral cloning plasmid carrying the genetic marker was constructed by genetic engineering technique.The recombinant virus vector carrying the genetic marker was saved and identified.The PCV1 iDNA marker Vaccine laid the foundation,the main research content is as follows:1?PCV2 Guizhou isolates and their bioinformatics analysisIn this study,the differences between the genotypes of PCV2 Guizhou isolates and the bioinformatics were analyzed.The aim of this study was to investigate the molecular genetic characteristics of PCV2 epidemic strains in Guizhou province.To construct the PCV1-2 chimeric virus The selection of genotypes and the prevention and control of PCV2.The genome size of PCV2 a,PCV2b and PCV2 d strains was1768,1767,1767 bp and the genome size of ORF2 was 702,702 and 705 bp,respectively,by comparing the collected PCV2 nucleotide sequences.The differencesamong the nucleotide sequences of PCV2 strains were mainly found in ORF2 and ORF1.The amino acid sequences of the same genotype strains were conserved and the genotypes of the isolates were different.The difference between PCV2 b and PCV2 d strain was the largest,and PCV2 b was different from PCV2 b.It was presumed that PCV2 b might be a kind of PCV2 a to PCV2 d.Transitional genotype strain;suggesting that PCV2 b strain is a good candidate vaccine strain for the development of PCV2 iDNA vaccine.2?PCV1-2m-V5 chimeric virus infection cloning plasmid construction and preliminary identificationAccording to the results of sequence analysis,the GZ-RH1 strain of PCV2 b type was selected as the parent strain.By introducing 1 V5-labeled primer,the V5 tag was introduced into PCV2 ORF2 by PCR.PCV1 ORF2 was used to replace PCV1 ORF2 and cloned into pcDNA3.1 eukaryotic expression vector.The recombinant plasmid pcDNA3.1-PCV1-2m-V5 was successfully constructed.The infected mice were inoculated into Kunming mice by intramuscular injection of the infected clones.The mice were collected at the 1st,2nd,3rd,4th and 5th week,respectively.The blood samples were collected by PCV1-2m-V5 The results showed that the test mice produced PCV1-2m-V5 viremia from the first week and continued until the third week.The positive PCR product was detected by the first week.The results showed that the sequence was Construction of pcDNA3.1-PCV1-2m-V5 infectious clone plasmid sequence is basically the same,indicating that through the mouse inoculation test,successfully rescued to obtain the PCV1-2m-V5 virus.Then,positive sera containing PCV1-2m-V5 virus and PCV2 GZ-RH1 virus were inoculated into PK15 cells respectively.V5-labeled monoclonal antibody and PCV2 positive serum were used as primary antibody,and the 5th generation virus-receiving cells were subjected to IFA detection The The results of IFA test showed that green fluorescence was detected in PK15 cells infected with PCV1-2m-V5 virus,and no significant fluorescence was observed in PK15 cells infected with PCV2 GZ-RH1 virus.The results of IFA test showed that the green fluorescence of PK15 cells was inoculated with PCV1-2m-V5 and PCV2 GZ-RH1.The 5th generation cell culture ofPCV1-2m-V5 and PCV2 GZ-RH1 virus was collected and subjected to Western Blot detection with the V5 tag as a primary antibody.The results showed that there was a specific purpose in cell culture of PCV1-2m-V5 virus The results showed that the rescued PCV1-2m-V5 virus could produce V5-tagged Cap protein in infected cells.Using this feature,the PCV1-2m-V5 virus could be used to amplify the cell culture of PCV2 GZ-RH1 virus.The serological method identifies the virus and wild virus infection.In addition,the ORF2-V5 gene was detected in the fifth generation cell culture of PCV1-2m-V5 and GZ-RH1 PCV2 virus by using the primer carrying the V5 tag,and the specific band was detected in the cell culture of the former Band,and in the latter can not detect the specific band,indicating that the pair of primers can be used to save the virus and wild virus to identify.PCV1-2m-V5 rescue virus electron microscopic observation of the results showed that in the V5 antibody immunoprecipitation of the experimental sample,can be seen with the size of PCV consistent with the size of the virus particles,indicating that the fifth generation of cell culture containing V5-labeled PCV1-2m-V5 particles.In this study,pcDNA1.1-PCV1-2m-V5 infectious cloning plasmid was successfully constructed and PCV1-2m-V5 rescue strain was successfully constructed.The results showed that PCV1-2m-V5 could proliferate in PK15 cells and the experimental design Specific primers can distinguish PCV1-2m-V5 from other PCV2 strains,and can immunodeactivate both V5 antibodies and rescue proteins against Cap protein,suggesting that ELISA with V5 protein The difference between the rescue and PCV2 wild-type antibodies was established,which laid the foundation for the development of PCV2 iDNA labeled vaccine.