Constructing The Non-Eseential Regions Of Swinepox Virus Isolated From Jiangxi And The Establishment Of Model Of PCV2 Infected Mice

Porcine Circovirus Type 2(PCV2)is a member of the circovirus family of the Circoviridae family.The PCV2 can cause a series of pig-related diseases,such as multisystemic degeneration syndrome and weaned sows in weaned piglets.Vaccine immunity is an effective means that prevent PCV2.Because there are few PCV2-negative pigs in clinical practice and the cost of swine animal models is high,this study selected mouse animal models to evaluate the immunoprotective effects of different PCV2 commercial vaccines.Swinepox Virus(SWPV)has the characteristics of a single host,a large genome,and a non-essential replication region.It can be used as a vaccine vector for genetic engineering.In this experiment,22 reading frames were randomly selected from 150reading frames of swinepox virus,and the gene deletion strains of the 22 reading frames were constructed by fusion PCR and homologous recombination methods respectively.Some of their biological characteristics were determined so that the screening could be used for high reproduction of non-essential regions for the expression of foreign genes.1.Establishment of PCV2 fluorescence quantitative PCR methodPrimers and probes were designed according to the sequence of PCV2 ORF1(Gen Bank accession number:MG744311.1),and a standard quality plasmid PUC19-TK1-ORF1-TK2 was constructed by restriction enzyme ligation to establish a fluorescent quantitative PCR system.The results showed that the reaction system of PCV2 fluorescence quantitative PCR was 50°C for 2 minutes,95°C for 30 s,95°C for10 s,60°C for 34 s for 40 cycles.The slope of the standard curve was-4.5,and the intercept was 47.76.The sensitivity can reach at 8 copies/?L,which has a 100-fold improvement over the traditional PCR method.It provides a PCV2 quantitative detection method for PCV2-infected mouse animal models.2.Establishment of Animal Model of PCV2 Infected Mice and Evaluation of Commercial VaccinesIn this experiment,6-8 weeks old female ICR mice were selected and intraperitoneal injection of PCV2 virus fluid had been set.Arranging different infections(10¹⁰,10⁹,10⁸,10⁷,0 copies/piece)and different slaughter time after infection(5d,10d,15d,20d,and 30d),the PCV2 virus content in lungs of mice was determined by fluorescence quantitative PCR method,and the optimal infection amount of PCV2 for ICR mice and the best slaughter time after infection were determined.The results showed that PCV2 had the best infection amount of 1×10¹⁰copies in 6-8 weeks old female ICR mice,and the lung PCV2 content was highest after 15 days of infection.PCV2 commercial vaccines A,B,C,D,and E were selected as evaluation subjects.ICR mice were injected intramuscularly in the leg and injected with 25?L of left and right legs.After 90 days,1×10¹⁰copies virus was injected intraperitoneally.After infection,the mice were slaughtered on day 15 and the virus levels of PCV2 in lungs of mice were measured using fluorescent quantitative PCR method.The immune protective effects of different commercial vaccines were compared.The results showed that different commercial vaccines for PCV2 had different protective effects on 6-8 weeks old of female ICR mice.Among them,vaccine A had the best immune protective effect on mice.The establishment of a PCV2-infected mouse animal model provides a viable assessment method for the evaluation of PCV2commercial vaccines.3.Construction of NER-deleted strain of swinepox virus Jiangxi isolateIn this experiment,22 reading frames were randomly selected from 150 reading frames of swinepox virus as a screening area for the replication of non-essential regions.The primers for the amplification of the left and right arms were designed based on the entire gene sequence of SWPV-JX20G strain.The left and right arms were amplified by the template of swinepox virus SWPV-JX20G strain,and the EGFP-P11-P28-ORF2 was used as an exogenous insertion gene.Twenty-two gene fragments were constructed by the fusion PCR method.Twenty-two strains of the NERV-deficiency virus were obtained by transfection and purification,and the PK15cells were infected.Obvious green fluorescence can be seen in infected PK15 cells,which indicates that the foreign gene EGFP has been stably expressed.The 22 genes are all replication non-essential regions of the swine pox virus SWPV-JX20G strain.4.Determination of biological characteristics of NER-deficiency virus strains of pig poxBy measuring the size of fluorescence spot of 22 strains of infected strains infected with PK15 cells,the pathogenicity of nursery pigs,the expression level of PCV2-Cap,and the effect of immune protection on ICR mice,the results were as follows:(1)22 deletion strains after infection with PK15 cells,100%fluorescence was observed,which indicates that 22 vaccinia NER-deficient strains can normally proliferate in PK15 cells;(2)Western blotting results showed that 22 strains of virus-deleted strains expressed differently in PCV2-Cap,and 11 strains of ORF014,ORF042,ORF052,TK,ORF099,ORF107,ORF121,ORF126,ORF142,ORF144,and ORF145 were highly expressed,and 7 strains of ORF008,ORF012,ORF015,ORF017,ORF038,ORF133,and ORF143 had low expression in PCV2-Cap;(3)The pathogenicity of pigs with different NER-deleted strains revealed that the ORF008,ORF121-deleted strains recovered faster when pigs were infected and the swinepox-spots were smaller.The deletion of ORF009 resulted in an enhancement of pathogenic ability to nursery pigs;(4)It was shown by measuring the protective effect of different NER-deficient strains on mice that the seven strains of ORF008,ORF009,TK,ORF107,ORF121,ORF141 and ORF143 were the most effective in protecting mice.ORF012 and ORF133-deleted strain had the worst protection effect on mice,and basically had no protective effect on mice.To sum up,ORF008,ORF121,ORF133 are suspected to be the virulence genes of swine pox virus,ORF014?ORF042,ORF052,ORF099,ORF107,ORF121,ORF126,ORF142,ORF144,ORF145 can serve as a high expression site for exogenous genes.