Preparation Of GP5 Protein Synthetic Peptide Antibody Of PRRSV SC Strain And The Research Of ADE

Porcine reproductive and respiratory syndrome is caused by porcine reproductive and respiratory syndrome virus which causing porcine reproductive disturbance, respiratory symptoms and high mortality as characteristics of animal infectious disease.Due to immunologic tolerance and antibody dependent enhancement (ADE), and variability is so strong, PRRSV is difficult to prevent in production. On the protein structure of PRRSV, GP5 protein is the main immunogenic protein of PRRSV,it can induce the production of neutralizing antibodies and has the strongest neutralizing ability to PRRSV. Meanwhile,it is the main reason causing the ADE, but the epitopes related with ADE and their function is not clear in the GP5 protein.For the research of PRRSV ADE, we used PRRSV SC strains to research as follows:1. Preparation and identification of PRRSV SC strain GP5 protein synthetic peptide polyclonal antibodies.Using DNAStar software and IEDB online to predict and select B cell epitope of GP5 protein of PRRSV SC strain(GenBank:ABL60902.1),we choose amino acid sequence located to 36aa-48aa and 145aa-156aa of this protein (SC:SSHLQLIYNLTLC and LC:LLDTKGRLYRWR),to synthesize artificially, and coupling linked with keyhole limpet hemocyanin as complete antigen.Taking antigen of coupled protein SC and LC to immunize white New Zealand female rabbits and prepairing polyclonal antibodies, the purity of polyclonal antibody SC and LC were both above 99%. Identification by Western blot showed Synthetic peptide coupling protein SC and LC were approximately 90kDa, and no nonspecific bands were detected, so the polyclonal antibody SC and LC had good specificity and affinity.Agar gel immunodifusion showed that the titer of polyclonal antibody SC was 1:16,the titer of polyclonal antibody LC was 1:8.2. Preparation and identification of PRRSV SC strain GP5 protein synthetic peptide monoclonal antibodies.Taking antigen of coupled protein SC and LC to immunize The Balb/c female mice three times with the mixture of the adjuvant and incomplete adjuvant with equal amounts of the adjuvant, respectively.Fusing The spleen cells of mice with antibody of higher titers after immunization with SP2/0 cells,2 positive clones were detected by ELISA assay successfully, which were named SC and LC. After the detection by IFA, both green fluorescent signals could be observed. Through intraperitoneal injection of mice to induce ascites to expand production and purification of monoclonal antibody by purification, the antibody titer were both reached 1:12800. Synthetic peptide coupling protein SC and LC were approximately 90kDa, and no nonspecific bands were detected, so the polyclonal antibody SC and LC had good specificity and affinity. The 2 kinds of monoclonal antibodies belong to IgG1 subtype.3.The ADE research of PRRSV SC strains with gradient dilution of hyperimmune serumWe designed a pair of amplification primer at conserved region based on the PRRSV ORF6 gene sequence published in Genbank.After the establishment of the standard curve of fluorescence quantitative PCR, the standard curve equation:Y=-3.295X+35.766, and took this equation to detect the copies of virus which infected Marc-145 cell at different time,we drawed one step growth curve,the results showed copies of virus was the highest between 48-64h after infection,which is the best time to harvest PRRSV SC strain.Then we mixed gradient dilution degree of hyperimmune serum with 105.4TCID50 PRRSV SC strain for 1h,and added to Marc-145 cell to hatch and cultivate for 64h, then collected extracted RNA in each cell to do reverse transcription and took reverse transcription product to do fluorescence quantitative polymerase chain reaction.The results showed that varies dilution of health mice serum had no effect on the multiplication of PRRSV SC strain,when hyperimmune serum dilution degree<1:1600,the copies of virus increased with the hyperimmune serum dilution decreased gradually, indicating the hyperimmune serum is immune to PRRSV SC strain at this time and making a significant inhibitory effect, but hyperimmune serum at two dilution 1:1600 and 1:3200, Comparied with the two groups of viral copies in cell liquid,the former was higher than the latter.Comparied with different dilution degree of healthy mice serum groups, analysis result of statistics software showed difference reached extremely significant level when dilution degree was between 1:100-1:1600,and it had no significant difference at dilution degree of 1:3200.The result of indirect immunofluorescence assay was exactly the same with fluorogenic quantitative PCR, so when the hyperimmune serum antibody dilution was 1:1600, PRRSV SC strain may exhibit ADE.