Construction Of Porcine Phage Antibody Library And Screening Of Anti-PRRSV Antibody

Neutralizing antibodies can bind to antigens on the surface of the virus,thereby preventing the virus from adsorbing target cell receptors and preventing the virus from invading cells,which plays an important role in antiviral infection.Therefore,neutralizing antibodies is a kind of antiviral therapy preparation with very good application prospect.However,anti-viral neutralizing antibodies prepared using mouse monoclonal antibody technology have heterogeneity in humans,livestock and other animals,resulting in limited clinical applications.In recent years,with the development of antibody preparation techniques,preparation of human or animal-derived neutralizing antibodies has become a research hotspot.At present,significant progress has been made in the development of fully human neutralizing antibodies for various viruses such as human immunodeficiency virus,influenza virus,Ebola virus,and dengue virus,and it has also been found that natural antiviral neutralizing antibodies may exist in humans.Compared with studies on human neutralizing antibodies,the development of animal-derived neutralizing antibodies such as porcine is still in its infancy.Porcine reproductive and respiratory syndrome?PRRS?is a viral infective disease with the clinical manifestations of breeding disorder of pregnant swine,serious respiratory symptoms for piglets and growth retardation,etc caused by the porcine reproductive and respiratory syndrome virus?PRRSV?.PRRSV infection or the immunity can induce strong humoral immune reactions and the specific antibodies can be detected7-9 days after the infection normally,but these antibodies have no neutralizing capacity to the intrinsic and extrinsic virus and they can even strength the infection;the neutralizing antibodies usually appear after 28 days.Because the neutralizing antibodies are generated slowly and irregularly,it is very difficult to control the disease.Antibody library is such a forceful tool to screen the antibodies of different animals that the porcine phage repertoire was constructed in this research and the antibody of neutralizing capacity was also screened from it so as to research the antibody response of porcine to the PRRSV.It was found that the screened neutralizing antibodies were polyreactive antibodies through further research before researching its function in the process of virus invasion into cells in combination with proteins KRT8?Keratin 8?and MYH9?Nonmuscle myosin heavy chain II-A?.The detailed research contents are as follows:1.Construction of porcine na?ve antibody library and screening of anti-PRRSV antibody PBMCs?Peripheral Blood Mononuclear Cells?of negative swine for the PRRSV antibody was separated to extract mRNA and then genes of heavy-chain variable regions?VH?and light-chain variable regions?VL?were obtained through RT-PCR technological amplification;VH and VL were than assembled into a scFv segment by a flexible peptide gene through overlap PCR.Phasmid pComb3 XSS and scFv segment went through Sfi I restriction enzyme digestion,recovery,connection and then electroporation-competent cells to obtain the na?ve library of capacity 1.5×10⁸ and the correction rate to insert the segment size is 80%upon identification.It was found that the similarity of VH sequence was 63.3%⁹2.5%through sequencing and there was no the same sequence to indicate the good diversity of the antibody library.It was found that the distribution of the VH gene family for the na?ve library followed the rule reported in the literature that the most three gene families accounted for over 40%and the most six gene families accounted for over 70%compared with IMGT database and the VH genes in the na?ve library were distributed in 13 different gene families to further indicate the good diversity of the na?ve library.The reconstructed phage antibody was obtained through super-infection of adding the helper phage and then screened by purified virions to obtain four phage antibodies N-28,N-36,N-48 and N-67.The obtained antibody gene was connected to pET-22 b expression vector and the recombinant proteins were mainly expressed in the form of inclusion body upon identification;the capability for the four antibodies to neutralize the virus was determined upon denaturation and renaturation of the inclusion body and N-28 as well as N-67 has certain neutralizing capability.2.Construction of porcine PRRSV immune antibody library and screening of anti-PRRSV antibodies We firstly vaccinated four piglets of 30 days with the PRRSV commercialized vaccine before infected them with highly pathogenic PRRSV?WUH3?,two kinds of American epidemic strains?VR2385 and VR2549?as well as domestic isolated epidemic strain?NADC30L?.High titer neutralizing antibodies were generated upon eight immune or infection experiments,the neutralizing titers for the above four strains ranged from 1:63.5to 1:858.7.The PBMCs of the PRRSV hyper-immune swine was separated to amplify the antibody gene and the immune library of capacity 2×10⁷ was obtained through times of electro-transformation;the correction rate was 89.4%to insert the segment size upon identification.The similarity of VH sequence was 70.3%⁹3.5%through sequencing and no same sequence was found to indicate the good diversity of the immune antibody library;VH number of gene families reduced from 13 to 6 of the natural library to indicate that the use rate for the antibody gene was obviously changed of the experiment swine upon times of vaccination.Three phage antibodies I-2154,I-1320 and I-141 were obtained through enrichment and screening and then the antibody gene was connected to the pET-22 b prokaryocyte expression vector;the neutralizing capability for the three antibodies to the three viruses?WUH3,VR2549 and NADC30L?was determined upon expression and purification and the result indicated that the three antibodies all had certain neutralizing function to the three viruses,in which the neutralizing effect of the I-141 was the best.The sensitivity of the three viruses to the antibodies was different and NADC30 L was most sensitive to I-141.We further analyzed the inhibition mechanism of I-141 on NADC30 L to find that I-141 restricted the virus invasion instead of affecting the virus adsorption.3.Mass spectrum identification and functional research of scFV binding protein Firstly biotin-labeled N-28,I-141 were incubated with MARC-145 cell lysate infected or not infected with PRRSV,respectively.The ferrite bead marked with the streptavidin was used to separate the scFv binding protein,for which the mass spectrum identification was conducted then.The PRRSV WUH3 protein database was retrieved for the mass spectrum result and no virus protein was identified in the two inoculated groups;it was found that 30 kinds of protein could all be identified in the four experiment groups though protein analysis identification to remind us that N-28 and I-141 could be polyreactive antibody.We chose the two known proteins KRT8?MYH9 to exert important functions in the process of virus invasion into the cells as the research objects to confirm the polyreactivity of these two scFv and it was found that N-28 and I-141 could be interacted with KRT8 and MYH9 to confirm the polyreactivity of these two scFv through the immune co-precipitation experiment.Then these two proteins were disturbed or over-expressed in the PK-15CD¹⁶³cell to confirm that these two proteins participated in PRRSV infection and further researches indicated that the expression quantity of these two proteins was increased in the process of PRRSV invasion into cells.