Development Of Lentivirus Vectored-VHHs Library And Screening Of Its Genes Against Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea(PED)is an acute contagious enteric disease that caused by Porcine epidemic diarrhea virus and can cause serious damage to the swine industry,lead to severe dehydration,diarrhea and vomiting,which is the most harmful to the lactation piglet,whose infection rates and mortality rates are extremely high,restrict the development of the swine industry worldwide.But until now,there is no effective methods and measures response to PEDV global outbreak.Therefore,it is extremely urgent to find effective drugs to prevention and control PED.Previous studies found that PEDV can not only proliferate in Vero cells providing with trypsin,But also can highly proliferate in IEC cells,who is the target cells of PEDV infection,and also can induce obvious Cytopathic Effect(CPE).So IECs and Vero cells can be used as screening ideal cell screen the target genes for against PEDV.Present study aimed to build an immune nano-antibody library of peripheral blood monouclear cells(PBMC)using lentivirus and other related technologies,then to find the genes with anti-PEDV effect.Connecting the VHHs library acquired through SMART technology to the expression vector of lentivirus.The packaging plasmid was transfected into293T cells with other three auxiliary plasmids of lentivirus,then harvest an infectious lentivirus virus library.The stable cell lines with anti-PEDV effect were constructed by using the library to infect Vero cells and IEC cells,screening by PEDV infection.The target genes sequence was obtained byRT-PCR,and analysis was performed in NCBI BLAST database to obtain the genes that have anti-PEDV virus.Details are as follows:1.Reconstruction of theLentivirus expression vector for library construction.The lethal gene CcdB-Cm was amplified by Using pFPAV3-CcdB-Cm as the template.Restriction Enzyme cutting site XbaI and BgLII were added to the upstream and downstream of CcdB-Cm fragments by designing primers,the product of the lethal gene CcdB-Cm cutted with Xba I and BgLII enzyme can be connected with the product of pCD513B-1cutted with Xba I and BamH I enzyme,by which the lentivirus expression vector pCD513B-1-CcdB-Cm was obtained for the construction of VHHs library.The modified lentivirus expression vector pCD513B-1-CcdB-Cm was identified by cutted with XbaI and Not I,by which we can obtain a specific fragment of 8100 bp and a specific fragment of 1400 bp,which indicate that the recombinant vector was successfully constructed.Comparing with the commercial lentivirus expression vector pCD513B-1,the modified lentivirus expression vector pCD513B-1-CcdB-Cm could dramatically increase the efficiency of VHH cloning.2.The acquisition of the Lentivirus nano-antibody immune library.the Lentivirus nano-antibody immune library was success constructed through SMARTer TM technology,the transformation efficiency and titer of the Lentivirus nano-antibody immune library were 7.26×10~6 cfu/3?g and 1.97×10~9 cfu/mL,respectively,the percentage of library insertion diversity was more than 95%,which met the demand of library screening.This method provided a new technology platform to establish a high-throughput nanobody screening in eukaryotic cells.3.The establishment of IEC and Vero cell line for stable express VHHs protein.The correct reeombinant lentiviral-vector plasmid and three other packaging plasmid p LP1,pLP2and pLP/VSVG were co-transfected into 293T cells to obtain an infectious recombinant lentivirus,the titer of the lentiviruses was 5~10×10~7 TU/ml.IEC and Vero cell lines were infected with the infectious recombinant lentivirus,the infection rate of the lentiviruses library was about 50%via fluorescence microscope detection.After treating the infected cells with puromycin,The IEC and Vero cell lines that can stabilize expression of nano-antibody immune library protein were successful obtained through the Western blotting assays.4.Screening and functional verification of anti-PEDV genes.The viable cells with anti-pedv were screened by infecting different cell lines using the Porcine epidemic diarrhea virus CV777,the result showed only IEC cell lines that stable express VHHs gene survived.Obtained the target genes VHH-P with anti-pedv effect though extracting RNA,RT-PCR,sequencing.Randomly select VHH-P2 and VHH-P6 to verified anti-PEDV function.Results showed that overexpression of VHH-P2 and VHH-P6 can lengthen CPE appearing time in Vero cells after infected with CV777,and the virus titer decreased as well,demonstrated that VHH-P2?VHH-P6 could inhibit proliferation of CV777.Western blotting assays showed the relative expression of N protein in PEDV was reduced in IEC cells expressing VHH-P2 and VHH-P6,which was consistent with the virus titer result.