Preparation Of Monoclonal Antibodies Specific For Prrsv And Immunogenicity Analysis Of Prrsv

Porcine reproductive and respiratory syndrome (PRRS) is a infectious disease characterized by reproductive failure and piglet sow respiratory diseases which caused by porcine reproductive and respiratory syndrome virus (PRRSV).The ORF2～7 genes in PRRSV genome encoding the virus GP2,GP3,GP4,GP5,M and N protein. N protein has the strongest immunogenicity and the largest quantity in virus particles,also is the advantage structure protein and the preferred protein for diagnosing PRRS.In this study,we cultured PRRSV virus on the Marc-145 and got the virus solution.After identified by PCR and high speed centrifugation,we got purified PRRSV which was determined by UV-Vis spectrophotometer for protein content as 1.35mg/mL.Inactivated the purified virus,determined the optimum coating antigen concentration as 2.438μg/mL through ELISA,optimized the condition of the reaction,then established the indirect ELISA.Using purified PRRSV to emulsified with Freund's adjuvant equivalently,then immunized BALB / C mice by 110μg / dose,once in two weeks and five times altogether. subcutaneous for 3 to 5 times and detected by ELISA method till the Serum antibody titers up to 1:6400 or more,Intraperitoneal injection of 330μg pure antigen immunization,3 days later, the mice were taken sterile spleen, preparation of spleen cells,fuse the spleen cells and SP2/0 myeloma cell by 5:1,when fusion cell is covered with holes until 1/3, detection with using the indirect ELISA, positive clones cells. By limited dilution cloning of the positive cells, by SDS-PAGE and Western blot identified two stable antibody secreting anti-N protein positive cell clone, named AD10 and BC12. Clones cultured cell lines expanded, taking about with 4~6×106 cells into the peritoneal cell suspension, 12 days after the collection of ascites, using the indirect ELISA, Determination of antibody titer was 1:160.The purified PRRSV antigen was divided into two parts, one was dealing with formaldehyde, the other was not dealing with formaldehyde. The immunogen was emulsified with equal amount of Freund's adjuvant. Twenty mice were randomly divided into A, B, C, D four groups, five mice each group, immunized mice by 110μg / dose, A group and B group mice were immunized by processed and unprocessed antigens one time, C group and D group mice were immunized by processed and unprocessed antigens two time interval 7 days. Collecting mouse tail vein blood for every 7 days, respectively detect anti-PRRSV,GP5,M,N protein antibodies by indirect ELISA. The results show that four experimental groups of mice can produce anti-PRRSV,GP5,M,N protein antibodies, anti-N protein antibodies are created firstly,after immunization 14 day, the antibody titer reached 1:1280 after immunization 21 day; anti-M protein antibodies appeared after immunization 42 days, antibody titers reached 1:80, anti-GP5 protein antibodies appeared after immunization 49 days, antibody titers reached 1:80. Two immune antibody titers than one slightly higher.At the same time each serum collected by 1:2,1:4 and 1:8 dilution and did the cells neutralization test, the results show that the four experimental groups of mice immunized serum only detected 42 days after immunization in and antibody titer of 1:2, the results show that the weak immunogenicity of PRRSV, the body can not produce effective protection.