Preliminary Establishment And Study Of An Indirect ELISA Based On The Cloning And Expression Of PCV3 Cap Protein

Porcine circovirus 3(PCV3)is a newly discovered infectious disease pathogen in recent years.This virus is susceptible to pigs of any age,This disease can lead to multiple systemic inflammation,dermatitis nephropathy syndrome(PDNS),reproductive disorders such as miscarriage,stillbirth,mummified fetus,weak fetus.In 2016,Palinski and other researchers of Kansas State University discovered the virus for the first time on a swine farm with dermatitis nephropathy syndrome and reproductive disorders.The PCV3 genome is 2000 bp in length and has three open reading frames: ORF1,ORF2 and ORF3.ORF1 is 891 bp in length and encodes 297 amino acids.It is a replicase protein(Rep),which is related to the replication initiation of the virus.ORF2 is 645 bp in length and encodes 214 amino acids.It is a structural protein(Capsid),which is the main antigen of the virus.ORF3 encodes 231 amino acids and its function is unknown.Since 2017,China has detected PCV3 in sick pigs with clinical symptoms such as reproductive disorders,pneumonia and dermatitis in many provinces and cities.The virus is widely distributed in China,but there are few reports on the serological detection methods of the virus antibody,and there is no standardization.This study aims to establish a highly sensitive,specific,simple and convenient ELISA method,which can provide scientific and effective means for epidemiological investigation and antibody level detection,and can provide reference for standardization of future detection methods,it will have great significance to the prevention and control of the disease.This study designed primers based on the PCV3 Cap gene sequence registered by Gen Bank(MF318451.1).Using a positive sample from the Guangdong swine farm for amplification and sequencing,a Cap gene sequence was obtained,named GDPCV3-Cap.Compared with the 15 PCV3 Cap gene sequences at home and abroad from Gen Bank,the homology shared 98.1% to 99.5%,and it has the highest homology with Guangdong strain MH491030.1,which is 99.5%.Genetic evolution analysis showed that GDPCV3-Cap is on the same evolutionary branch as Guangdong strain MH491030.1,indicated that genetic relationship among these two strains is closer.The results of amino acid locus analysis showed that the sequences obtained in this experiment were only mutated at positions 24 and27,and the amino acid sequence differences between strains in different regions were small.In this study,we used the obtained sequence,truncated the signal sequence,then connected to p ET-30 a vector after codon optimization and whole gene synthesis,successfully constructed p ET-30a-PCV3 Cap prokaryotic expression vector.Transformed the expression vector into BL21(DE3)competent cells and induced to express for 16 h using IPTG at a final concentration of 0.1 mmol/L at 15℃,SDS-PAGE showed that the molecular weight of the expressed protein was around 23 k Da,which was in agreement with expectations.The protein was purified by Ni-IDA agarose gel.The SDS-PAGE showed that the band was single,the purity was above 90 %,and the concentration was 0.356 mg/m L.The Western-blot identification showed that the purified protein had good antigenicity.Using the successfully expressed and purified PCV3 Cap protein as a coating antigen,Through the selection and optimization of each reaction condition,an indirect ELISA method for detection of PCV3 serum antibody was established,and final conditions are: The antigen coating concentration was 1 μg/m L,and the coating conditions were incubated at 37℃ for2 h and then coated at 4℃ overnight.The blocking solution was 5% skim milk powder,and the blocking condition were incubated at 37℃ for 2 h and then at 4℃ overnight.The serum(primary antibody)dilution was 50-fold dilution,and the Serum incubation conditions were incubated at 37℃ for 90 min.The dilution ratio of the secondary antibody is 10000 times,and the condition were incubated at 37℃ for 60 min.The color development condition are at room temperature and kept away from light for 15 min.After identification,the ELISA method established in this study has no cross-reaction with the positive serum of PCV2,PRV,PRRSV,CSFV,PEDV and PPV,indicating that the specificity is strong.Repeatability tests showed that the intra-assay coefficient of variation was between 1% and 5.8%,and the interassay coefficient of variation was between 3.3% and 8.4%,indicating good repeatability.Sensitivity tests showed that positive samples were still detectable when the serum was diluted to 6400 times.Using the ELISA method established in this study,a total of 834 sera from different farms in various provinces between 2018 and 2019 were detected.The results showed that521 were positive,and the overall positive rate was 62.47%.