Molecular Epidemiology Investigation Of Porcine Circovirus Type 2 In Henan And Research Of Its Affinity Ligand

Porcine circovirus type 2(Porcine circovirus type 2,PCV2)is the pathogen of porcine circovirus associated diseases(Porcine circovirus-associated diseases,PCVAD).It is one of the main pathogens in global pig industry,which has brought huge economic losses to the global pig industry.At present,vaccine immunization is the main means to prevent PCVAD.Although a variety of vaccines have been on the market since 2009,from whole virus inactivated vaccines to subunit vaccines,the infection rate of PCV2 is still high.In view of this phenomenon,we put forward two questions.Firstly,PCV2 vaccines are mainly divided into two categories now,PCV2 whole virus inactivated vaccine and PCV2 subunit vaccine.The genotype of PCV2b is dominant,and the remaining is PCV2a.If the prevalent PCV2 genotype of in field does not match vaccine genotype,the effect of the vaccine may be reduced or lost.So it is necessary to investigate the molecular epidemiology of PCV2.Secondly,the effective components of PCV2 vaccine are inactivated PCV2 virus particles and virus-like particles(VLPs),the content and purity of which directly determine the quality of the vaccine.In vaccine production,the purified PCV2 virus particles and VLP with high concentration are still difficult to obtain,at the level of primary purification(concentration,precipitation and centrifugation).Because of the purification effect is poor and unstable,the obtain vaccine can cause side effects and finally waste animal immune potential.At present,a rapid and efficient purification method is urgently needed.Based on the above two questions,the molecular epidemiology of PCV2 in Henan province from 2015 to 2018 was investigated,and the affinity ligands of PCV2 were prepared and selected,which provided technical support for the prevention and control of PCV2 and the purification of vaccines.This study is divided into three parts:1.Molecular epidemiology investigation of PCV2 in Henan province from 2015 to 2018This study investigated 117 clinical samples from Henan Province from 2015 to 2018,and found that the infection rate of PCV2 was 62.4%(73/117).The genomes of PCV2 in 37 positive clinical samples were amplified and sequenced.Phylogenetic analysis based on PCV2 ORF2 and full genome revealed that the all 37 strains belonged to PCV2a(3/37),PCV2b(21/37)and PCV2d(13/37),suggesting that PCV2b and PCV2d were the prevalent genotypes during this period.The amino acid sequence alignment of PCV2 Cap protein showed that eight amino acid alteration sites(Y8F,F53I,157V,A68N,S121T,T134N,S169G/R and V215I)were found in PCV2d ORF2 protein.According to the location of these sites on the Cap protein,we speculate that these changes may cau se the immunoge nicity chan ge of Cap protein.In addition,the selective pressure analysis showed that there were five positive selection sites(30,63,89,90 and 190)in PCV2 ORF2,and the changes of these positive selection sites might be beneficial to the virus escape from the host immune system.2.Preparation and preliminary application of PCV2 nanobodyIn this study,soluble PCV2 nanobodies were successfully expressed in the periplasm of E.coli.High purity PCV2 nanobodies were obtained by Ni affinity chromatography and SP cation exchange chromatography.Colloidal gold test paper and IFA proved that it could bind to PCV2.The affinity constant was KD=2.6×10-6M determined by SPR.When the concentration of PCV2 nanobody was 2 mg/mL,the coupling efficiency was 92.3%and the ligand loading was 1.846 mg/mL,which was the best condition for preparing affinity adsorbent.The pH of elution condition was determined to be 2.2.Affinity adsorbent can be used to purify PCV2 culture solution.The purity of the product is high identified by SDS-PAGE and WB.The complete virus particles can be observed by transmission electron microscopy.This study provides a way and reference for vaccine enterprises to purify PCV2 virus particles(or VLP)in the future.3.Panning of peptide ligands of PCV2In this study,PCV2 peptide ligands were screened from a random 12-peptide library displayed by a bacteriophage.In the course of the research,two strategies were tried,one used PCV2 Cap protein as the target,the other selected PCV2 virus as the target.In strategy 1,six kinds of bacteriophages were obtained after three rounds of selection,of which the bacteriophage C2(YHDCFSAGFCIG)was highly enriched.In strategy 2,three kinds of bacteriophages were obtained after four rounds of selection,of which the bacteriophage CX4(WFPTGATRLVV)was highly enriched.After synthesized the corresponding peptides,ELISA results showed that only peptide C2 and C12 could bind to the PCV2 Cap proteins,while all the selected peptides couldn't bind to PCV2.When peptide C2 and C12 were coupled with agarose beads at 2 mg/mL,the coupling efficiency was 85.2%and 85.9%,and the ligand loading was 1.704 mg/mL and 1.718 mg/mL.The adsorbent prepared has a certain adsorption effect on PCV2 Cap protein.