Screening And Application Of PCV2 Specific Single Domain Antibody And The Research On The Interaction Of PCV2 With Porcine Alveolar Macrophage

Porcine circovirus type 2（PCV2）is a contagious pathogen threaten the swine industry worldwide.Sub-clinical infection is the main feature of PCV2 infection and most of PCV2-infected pigs have no clinical symptoms.Thus,it is important to develop a sensitive and specific diagnostic method for the prevention of PCV2.The single domain antibody（sdAb）is the variable region of the heavy chain antibody derived from the camel.It is currently known the smallest antibody,and can be obtained by recombination expression in vitro,which make them has promising application in the study of the pathogen-host interaction and the pathogen diagnosis.The typical marker in PCV2-affected pigs was immunosupression,which may associate with the ability of PCV2 to infect immune cells.Porcine alveolar macrophage（PAM）is a target cell of PCV2,which play an important role in PCV2 infection and transmission.In this study,the pathogenic diagnostic methods of PCV2 were developed based on sdAb.Meantime the mechanisms of PCV2 interaction with PAM were researched by the analysis of differential expression of the inflammation correlation factors and the TGF-βsignaling targets in PAM post PCV2infection using PCR array method.The studies were as follows:1.A sdAb library was constructed from PCV2 vaccine immunized C.bactrianus and three sdAbs（sdAb-c1/c3/c4）specific to the capsid protein（Cap）of PCV2 were selected and expressed.sdAb-c1/c4were specific to PCV2 Cap,but sdAb-c3 shows binding activity with both PCV1 and PCV2 Cap,which was determined by an enzyme linked immunosorbent assay（ELISA）.The sdAb-cs were able to binding with different epitopes in PCV2 Cap that was showed by an additive ELISA.Additionally,sdAb-c1/c3/c4 showed association rate constant of 1.84×10⁵M^-1 s^-1,5.49×10⁴M^-1 s^-1,1.46×10⁵M^-1s^-11 and dissociation rate constant of 9.00×10^-3 s^-1,9.91×10^-3 s^-1,1.18×10^-3 s^-1 respectively,which was analyzed by surface plasmon resonance（SPR）.Our study confirmed that sdAb-cs with high affinity and specificity to PCV2 Cap have the potential application for the diagnostic development of PCV2.2.psdAb-c1 was expressed as fusion with an alkaline phosphatase（AP）.The binding activity and specificity of psdAb-AP fusion was similar with psdAb,but the functional affinity of psdAb-AP fusion was about 5 times greater than psdAb(psdAb,KD=4.94×10^-8/psdAb-AP,KD=9.47×10^-9)as determined by SPR and an ELISA.As a indicator,psdAb-AP fusion could detect PCV2 Cap down to 0.01μg/lane in Western blot assay and 0.05μg/mL in a direct ELISA.Compared with indirect fluorescence assay（IIF）,immunocytochemistry assay（ICC）based on the psdAb-AP fusion was more sensitive and efficient which need less operation steps and shorter time.3.The psdAb-c4 was expressed as fusion with green fluorescent protein（GFP）and red fluorescent protein（RFP）and conjugated with quantum dots（QDs）respectively.The label of GFP/RFP/QDs did not affect the binding activity and specificity of psdAb which were detected by ELISA,Western blot,and tissue adsorption assay.PCV2 in cells can be visualized using GFP/RFP/QDs-psdAb probes,which was consistent with the results of IIF,transmission electron microscopy（TEM）,and qPCR assay.However,compare with GFP/RFP-psdAb,QDs-psdAb was more suitable as a visualization probe of PCV2 due to the optical properties of high stability and reduced photobleaching.4.An immunomagnetic nanobeads（IMNBs）functionalized with psdAb-c1 was developed successfully.SDS-PAGE,ELISA,TEM,and particle diameter analysis showed that PCV2 can be captured by IMNBs.And approximate 2.57±0.13 mg of PCV2 Cap or 0.97±0.064×10⁶ copies of PCV2 can be capured by1mg of IMNBs in 30 min showed by capture efficiency assay.A fast diagnostic method for PCV2 was developed based on IMNBs and QDs-psdAb probe,which show high sensitivity with the detection limit as low as 10³ copies/mL of PCV2.The detection of clinical samples using this method indicated that it is promising for the rapid and effective detection of PCV2 in complex clinical samples.5.The expression profile of inflammation correlation factors and TGF-βsignaling targets in PAM post PCV2 infection were analyzed using PCR array method.Inflammation associated chemokines（such as CCL2,CCL3,CCL4,CCL5,CCL8,and CXCL2）,colony stimulating factors（such as M-CSF,GM-CSF,and G-CSF）,and cytokines（such as IL-1α,IL-1β,IL-8,IL-10,IL-33,and TNF-α）were up-regulated significantly（P<0.05）in 1hpi in mRNA level.And 63（63/84）of the TGF-βsignaling targets were differential expressed（P<0.05）at different time post PCV2 infection（1 hpi,24 hpi,and 48 hpi）.These finding provide a basis for revealing the mechanisms of interaction between PCV2 and PAM.