Construction Of PCV3 Infectious Clone Plasmid And Virus Rescue Stud

Porcine circovirus type 3(PCV3)is a new type of circovirus associated with acute porcine dermatitis and nephrotic syndrome(PDNS)-like clinical symptoms.The genome size of porcine circovirus type 3(2000bp)was identified by macrogenomic sequencing.Since the discovery of PCV3,the virus has been reported in domestic pigs around the world.The clinical and pathological manifestations related to its infection are thought to include PDNS,reproductive disorders,diarrhea,systemic inflammation,respiratory diseases and central nervous system diseases.The high incidence of PCV3 may pose a potential threat to the global pig industry,but the reasons for the emergence and spread of PCV3 are still unknown.Virus isolation and culture is also a very difficult threshold for in-depth research.At present,the prevention and control of PCV3 is still a difficult problem.The acquisition of PCV3 strain is the key to the preparation of related vaccines and the development of corresponding antiviral drugs.In this study,the infectious clone plasmid of PCV3 was constructed by reverse genetic technique of circovirus,and the virus rescue was studied in vivo and in vitro,and the conditions of prokaryotic expression of PCV3 Cap protein were optimized in order to lay a foundation for the research of PCV3 etiology,pathogenic mechanism and new vaccine.The main research contents are as follows:1.Construction of PCV3 infectious clone plasmid and virus rescueIn order to obtain the PCV3 rescue strain and facilitate the further study of the virus,two pairs of specific primers were designed to amplify the virus genome from the PCV3 circular genome,and the virus genome was cloned into pMD19-TSimple Vector to construct a recombinant plasmid.The target fragment was digested by double enzyme and subcloned into pcDNA3.1(+)eukaryotic expression vector.The infectious clone plasmid pcDNA3.1(+)-PCV3 was successfully constructed by sequencing.The infectious clone plasmid was transfected into PK-15 cells mediated by calcium phosphate.72 hours later,the total RNA,RT-PCR of the transfected cells was extracted.The results showed that there was transcription of PCV3 Cap gene in the transfected cells.At the same time,the transfected cells were subcultured according to synchronous inoculation and routine inoculation,respectively.After receiving the virus,the nucleic acid of cytovenom was extracted and the PCV3ORF2 gene was detected by PCR.The results showed that the target band of 645 bp could be detected in the 1st and 4th generation of cell venom,and the results after the fourth generation were negative.The weak target band could only be detected in the first and second generation cytovenom after routine exposure.In order to further evaluate the infectivity of the PCV3 infectious clone plasmid in Kunming mice,10 mice were randomly divided into two groups.Each mouse in the experimental group was intramuscularly injected with 200μg pcDNA3.1(+)-PCV3 infectious clone plasmid,and each mouse in the control group was injected with200 m L normal saline.Blood samples were collected on the 7th,14 th,21th and 28 th day after inoculation,and the serum was taken to detect the duration of PCV3 viremia.The results showed that viremia could be detected in 4 Kunming mice at 7 days,and the viremia lasted up to 21 days.In order to further verify the infectivity of the rescue virus,the sera of mice positive for 7 days were inoculated into PK-15 cells and cultured continuously,and the PCR detection of Cap gene in each generation of cytovenom was carried out.The results showed that PCV3 rescued by mice could proliferate in PK-15 cells,but the ability of proliferation was weak.After continuous passage to the 8th generation,the result of Cap gene PCR detection was negative.The results showed that the pcDNA3.1(+)-PCV3 infectious clone plasmid constructed in the experiment was infectious to PK-15 cells and mice,and PCV3 could be obtained to save the virus after transfection or in vivo rescue in mice.However,the ability of passage and proliferation of PCV3 rescue virus in PK-15 cells is weak,which is not suitable for routine inoculation culture of PK-15 cells.The methods of virus proliferation and passage need to be further studied and optimized.2.Optimization of prokaryotic expression of porcine circovirus type 3 Cap proteinIn order to verify the identification results of PCV3 saving virus,it is necessary to prepare anti-PCV3 monoclonal antibodies.At present,the Cap protein of PCV3 prepared in our laboratory is mainly expressed in the form of inclusion body.although the expression is high,the purification of inclusion body is more tedious,which brings some difficulty to the preparation of antibody.In order to obtain PCV3 soluble Cap protein that can be highly expressed,the induction conditions of the constructed prokaryotic expression plasmid pET-32a(+)-PCV3-Cap were optimized.On the basis of the original conditions of IPTG final concentration 1 mmol/L,induction temperature 37℃ and induction time 4 h,and referring to other literature reports,the optimization study was carried out by reducing the induction temperature and reducing the amount of IPTG,and the fusion expression protein was verified by SDS-PAGE gel electrophoresis and Westernblot test with His tag antibody.The results showed that when the final concentration of IPTG was 0.1 mmol/L,the induction temperature was 20℃ and the induction time was 20 h,the soluble Cap protein of PCV3 could be efficiently expressed.This study laid a foundation for the preparation of PCV3 antibody,the establishment of serological differential diagnosis and the development of genetic engineering subunit vaccine.