Identification Of The Functional Domain Of The Receptor Papn From Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in piglets that leads to great economic losses in East Asia. It belongs to group I coronavirus.There are limited research of contributioin for pAPN and mechanism of viral entry.In2007, it is confirmed that pAPN was a functional receptor for PEDV. Afterwards, there is another result to prove that the infection rate of PEDV increasingly correlateing to the density of pAPN.In2010, Indifferent concentrations of pAPN was added in Vero cells before inoculating virus, and adding pAPN in different ways,plague assays showed pAPN could increase number of virion which attached to cells directly.Up to now,ther was no research for the contribution of pAPN on PEDV propagation.To investigate which part of pAPN is the fuctional domain for PEDV,we segmented the pAPN without signal peptide into three fragments.then used a pair of isocaudarner-Xhol I and Sal I connecting the SPA, SPB and SPC to signal peptide and constructed them to pFastBacTM1respectively,and expressed them in BAC to BAC system. The results indicated that we got42kD SPA protein,56kD both SPB and SPC protein identified by SDS-PAGE and Western-blot,they all matched what we expected.Then we immunized mice with the three kind of protein to preparing polyclonal antibodies.Three kind of protein could all ineracting with the natural pAPN,and proved that the protein we made had some specificity.To expressing the three fragments of pAPN.we constructed to pcDNA3.1again and transfected them to the Nonpermissive cells—MDCK of PEDV and identified whether SPA,SPB and SPC were expressed by immunofluorescence.The result shows that ineracting with the anti-serum of pAPN SPA.SPB and SPC all appeared bright green fluorescence and that means SPA,SPB and SPC were all expressed in MDCK. After the PEDV infected the MDCK which expressed SPA,SPB and SPC respectively48hours,we observe the lesion on MDCK,and found that MDCK which expressed SPC showed a distinct lesion:a lot of cells were dropped and modified.Meanwhile,we used immunofluorescence to test that MDCK which expressed SPC can be found green fluorescence clear and bright,and that illustrated SPC interacted with PEDV further.Infected the PEDV in the MDCK which expressed SPC for five generation and the PEDV can be tested by PCR.This also proved that spc which expressed in MDCK can proliferate the PEDV.Finally, we added the antiserum of SPC to the MDCK which expressed SPC and infected with PEDV,the results certify that the lesion can be controlled.all the results demonstrate that spc plays an important role in PEDV invading into cells and it is the functional part of pAPN.And from this research we finished the selection of functional domain of pAPN for the first step,and lay a foundation for the multiplication of PEDV in nonpermissive cells.