Construction And Activity Identification Of Chimeric Single-chain Fragment Variable Antibody Against Porcine Epidemic Diarrhea Virus

Currently,commercial vaccines to prevent the porcine epidemic diarrhea are mainly attenuated vaccines and inactivated vaccines.Since the winter of 2010,PED in our country erupted again,however,the available commercial vaccines failed to protect the disease.Because of incomplete immune system in new piglets,it is difficult to stimulate the active immunity reaction.Then,it may be a better option to develop new recombinant antibodies for passive immunization by genetic engineering technology.Based on the former development of PEDV hybridoma 2D1,total RNA is extracted and reverse transcription PCR is conducted to get a full set of c DNA.Then,heavy chain variable gene and light chain variable gene is amplificated with the 17 and 19 primers respectively.We screened 2 VH genes(VH5 and VH8)and 1 VL gene sequences and specific primers for variable fragment amplification.Kunming mice were immuned with PEDVImmuned kunming mice with PEDV S1 protein expressed in prokaryotic to get anti-S1 serum,and total RNA is extracted and reverse transcription PCR is conducted to get c DNA.Heavy chain variable gene and light chain variable gene with specific amplification primers were amplificated.One of the clones matched completely with VH5 gene sequence,and one of the ones matched 97.18% with VL gene sequence.Then we could confirm the variable fragment gene of single chain antibody against PEDV.Total RNA from porcine spleen is extracted and reverse transcription PCR is conducted to get c DNA,and Fc gene of pig is amplificated with specific primers.Single-chain variable fragment(scFv,765bp)and scFv-Fc(1128bp)were amplified with SOE-PCR,and recombination protein p EASY-scFv and p ET28a-scFv-Fc were constructed and expressed in prokaryotic system.Molecular weight of scFv and scFv-Fc were identified as 32 KDa and 45.8KDa respectively by SDS-PAGE,Western-Blot and Elisa.Finally,scFv and scFv-Fc were identified by using HRP anti-labeled 6×His and HRP goat anti-swine.The results showed that scFv and scFv-Fc had binding activity with PEDV,and scFv-Fc protein could be recognized by HRP goat anti-swine.Therefore,our research laid the theoretical foundations for PEDV recombination protein,and had a more profound significance in practical application with using Fc to modify scFv.