The Capsid Protein Expression And The Construction Of Infectious Clone Of Porcine Circovirus Type 3

Porcine circovirus type 3 is a novel circovirus which was first reported in 2016 and it can cause porcine dermatitis and nephrotic syndrome(PDNS),reproductive failure,and cardiac and multi-systemic inflammation.The PCV3 genome is 2000 nt in length and it has three major open reading frames(ORF),ORF1 encodes the replicase protein,and ORF2 encodes the capsid protein.The homology between PCV3 and PCV2 genome is only 50%,and the homology with PCV1 genome is only 37%.In this experiment,the prokaryotic expression system and eukaryotic expression system(swinepox virus vector)were used to express PCV3 capsid protein.In addition,the virus was obtained by infectious clone,which lays the foundation for the establishment of PCV3 immunological detection method,and biological characteristics research.1.PCR detection of porcine circovirus type 3According to the ORF2 gene sequence of porcine circovirus type 3(Accession number: KY354031.1)in Gen Bank,the porcine circovirus type 3 PCR detection primers JC9F/R was designed,and the PCR detection method of PCV3 was established.PCV3 PCR was performed on 181 porcine tissue samples from September 2018 to May 2019 in Jiangxi Province by this PCR detection method.Five PCV3 positive cases were detected,and the positive rate was 2.76%.2.Prokaryotic expression of PCV3-ORF2In this experiment,the ORF2(645 bp)of porcine circovirus type 3 Accession number: KX458235.1 was used as a reference sequence to design the PCV3-ORF2 primers.The PCV3-positive disease DNA was screened by clinical disease screening,and the PCV3-ORF2 fragment was amplified by PCR and cloned into p ET-32 a and p GEX-4T-1 respectively to construct the recombinant expression vector p ET-32a-PCV3-ORF2 and p GEX-4T-1-PCV3-ORF2.Then they were transformed into BL21(DE3)p Lys S host bacteria,respectively.After induced expression,the protein expressed by p ET-32a-PCV3-ORF2 strain didn’t appear 45 k Da target band in SDS-PAGE;However,the protein expressed by p GEX-4T-1-PCV3-ORF2 strain didn’t appear 50 k Da target band.It was determined that the PCV3-Cap protein was not expressed.3.Eukaryotic expression of PCV3-ORF2The swinepox virus transfer vectors which expressing PCV3-Cap protein were constructed,and P28-PCV3-ORF2-His was amplified by PCR using PCV3-positivedisease DNA as the template.The fragment was ligated into the p SWE101 vector by the In-fusion method.The swinepox virus Jiangxi isolate(SWPV-JX20G)was used as the parental virus,and the TK gene was used as the insertion site of the foreign gene,and Vaccinai virus(Vaccinai virus,VV)late promoter P28 was used as a promoter of PCV3-ORF2 to construct recombinant transfer vector p SWE101-283 CH.The constructed plasmid was transfected to produce a recombinant swinepox virus expressing the target protein,and the recombinant protein was purified.The results showed that the sequencing results of recombinant swinepox virus TK locus of were consistent with the expected results.SDS-PAGE identified the expression of the target protein at 26 k Da,and the PCV3-Cap protein was successfully expressed by the swinepox virus vector,which provided a basis for further studying on the immunogenicity of PCV3-Cap protein and establishing PCV3 antibody detection methods.4.Construction of PCV3 infectious clonesThe PCV3 genome reference sequence(KX458235.1)was used as a template,and the full-length plasmid of PCV3 was synthesized using the Bam H I from the 21 th base of the PCV3 genome.Using this plasmid as a template,and the PCV3 full-length primer P3001F/R carrying Bam H I restriction site was used to obtain full-length DNA of PCV3 by PCR,and then PCV3 loop DNA was constructed by Bam H I digestion and T4 ligase linkage.The cohesive ends were supplemented with two Bam H I restriction sites by upstream and downstream primers,and then PCV3 circular DNA was constructed by Bam H I digestion and T4 ligase ligation,and PK-15 cells were transfected.The T4 ligation product was verified by PCR of DNA cyclization verification primers,and it was confirmed that the first and last consecutive PCV3 genomic DNA was present in the T4 ligation product.After the circular DNA was transfected into PK-15 cells using liposome,the passage was blindly transmitted for 19 passages,and no cytopathic effect was observed.The DNA of rescued virus cell culture was extracted and PCR detection of PCV3 nucleic acid was performed.The detection result of each generation of rescue virus was PCV3 positive,and it was determined that the virus rescue of PCV3 was successful,and the PCV3 virus was successfully rescued by the infectious cloning method.