Analysis Of The Function Of PCV2 Rep Promoter And Construction Of Infectious Clone

【Objective】Porcine circovirus contains two serotypes,PCV1 and PCV2.Some kinds of diseases are related with PCV2 in porcine groups.Especially,PMWS is the most serious and has made great economic losses in the word.So,researching the mechanism of pathogenesis of PCV2 is very significant for prevention and treatment of diseases which are related with PCV2.In this reseach,PCV2-DT was going to be isolated,and Rep and Cap promotors of PCV2 was going to be located,and the function of vISRE which lies in Rep promotor was going to be analyzed,and infectious clone of PCV2 and recombinant virus of PCV2 with HA-Tag were going to be constructed.This research would lay a foundation for studying multiplication in vitro,pathogenicity of PCV2, pathogenic mechanism,vaccine development,molecular diagnosis.【Methods】(1) PCV2 was isolated from tissue of pig which came from DT city and died of PMWS by using PK-15 cell,and its genomic sequence was analyzed.Piglets of 42 days which were free of PCV1,PPV,PRV,PRSV were inoculated with PCV2-DT virus.(2) PCV2 promotors were predicted by using promoter prediction software and the sequences predicted were amplified by PCR and cloned into pGL3-Basic to construct pGL3-Bp1,pGL3-Bp2,pGL3-Bp3,pGL3-Bp4 as well as pGL3-Bp5 which contains known PCV1 Rep promoter.All recombinated plasmids were transfected into PK-15 cell,and the activity of promoters was detected by using Luciferase Assay System.(3) PGL3-Bp2 which contains PCV2 Rep promoter and PGL3-Bp5 which contains PCV1 Rep promoter and PGL3-Bpm which was constructed through mutating PCV2 Rep promoter in vISRE were respectively transfected into PK-15 cell.After being stimulated by different doseα-IFN,the changes of activity of promoters were detected.(4) Two copy gene of PCV2 was tandemLy cloned into pSK(+) to construct pSK-2PCV2.And pSK-2PCV2 was transfected into PK-15 cell,after 4 passages,the infected cell was detected by IFA and RT-PCR.(5) HA-Tag gene was cloned into Cap gene end by SOE-PCR.The PCV2 gene with HA-Tag was cyclized and amplied Then,circo-DNA-HA was transfected into PK-15 cell.After 4 passages,the cell was detected by RT-PCR,IPMA,Amplifying corresponding fragment and sequencing,HA experiment.Then,the stability of HA-Tag of the recombinated virus was detected by restriction enzyme NcoⅠdigestion.【Results】(1) PCV2-DT is succeedly separated.Comparing with 35 domestic and abroad strains,the homology of nucleotide sequence are all over 95%,and only some sites mutated The phylogenetic tree demonstrates that there is no obvious regionalism between different strains of PCV2.After being inoculated with PCV2-DT for10 days,the piglets emerged the symptoms of PMWS.(2) PCV2 Rep promotor is located in 1705nt-1968nt;PCV2 Cap promotor is located in 834nt-1309nt.The activities of Rep and Cap promoter are more stronger than control SV40 promotor.(3) The activities of promotors are inhibited byα-IFN in different extent.The activity change of PCV2 Rep promoter is positively correlated with the dose ofα-IFN.But different doses ofα-IFN had no obvious influence on the activity of PCV1 Rep promoter.After vISRE being mutated,the activity of PCV2 Rep promoter weakens and becomes sensitive toα-IFN.Whether mutated or not,the activities of promotors were heavily inhibited by high doseα-IFN.(4) PCV2 is detected in PK-15 transfected with PSK-2PCV2 for 4 passages by RT-PCR and IFA.(5) Recombinant virus with HA-Tag(PCV2-HA) is detected in PK-15 transfected with Circo-DNA-HA for 4 passages by RT-PCR and IPMA,but PCV2-HA has no activity of adsorbing rhodocyte of chicken.【Conclusions】(1) The homology between PCV2-DT and other 35 domestic and overseas strains was very high.PCV2-DT,and exists some sites mutated,and could induce piglet to emerge PMWS.(2) Rep and Cap promotor of PCV2 were firstly located by using luciferase as reporter gene(3) The activities of PCV promoters could be inhibited byα-IFN.Andα-IFN possibly regulates replication of virus wereby vISRE.(4) PSK-2PCV2 has infection activity,transfected into PK-15,could produce PCV2 virus.(5) Circo-DNA-HA has infection activity,transfected into PK-15,could produce recombinated PCV2 virus with HA-Tag.