Optimization Of A Protocol For Propagation Of PRRSV-resp Strain And Preparation For Its Hyperserum And Monoclonal Antibody

Porcine reproductive and respiratory syndrome virus (PRRSV) resp-strain which is liable to result in typical cytopathic effect (CPE) is one of the most widely used vaccine strains, and it has been widespread applied to produce attenuated or inactivated vaccine of PRRS at present. But the propagation of PRRSV-resp strain on Marc-145 cell is affected by many factors and its titer hasn抰 always been stable for a long time, which is an obstacle to the production and application of PRRS vaccines. This thesis thoroughly studied and optimized the factors of the propagation of PRRSV-resp strain on Marc-145 cell, which had laid a good basis for large-scale production and application of PRRS vaccines. The experiment showed that taking the PRRSV-resp strain titer as the standard, the optimum fit for PRRSV propagation was as follows: cell concentration was 100,000-150,000 per ml; the medium was MEM plus 8% of fetal bovine serum; the PH of the medium ranged from 7.0 to 7.4; the inoculation dose of PRRSV was 200TC1D50 per nil; the absorbtion time was 30 minutes at 370C; the harvest time was 72-96 hours after virus inoculation; the virus needed to be freeze-thawwed 3 times at -20 0C after harvesting.Cyclophosphamide pretreatment technique is often used to prepare for the antibodies of unknown or rare antigens in complex tissues, but it is hard to master2the injection dose and immune procedure of cyclophosphamide. This puzzle was solved in the article, and hyperserum with high purity against PRRSV-resp strain by rabbit has been successfully produced with cyclophosphainide pretreatment technique. hi this experiment, the tested rabbits were pretreated with normal Marc-145 cell components and cyclophosphasnide, then vaccinated with inactivated and oil-emulsified porcine reproductive and respiratory syndrome virus. The neutralization value of the hyperserum against PRRSV obtained by rabbit was 1:32 and its ELISA value was 1:640. The hyperseruni set up a basis for purifying and detecting PRRSVIndirect enzyme-linked immunosorbent assay (I-ELISA) and indirect fluorescent antibody (IFA) were developed to detect monoclonal antibodies (McAb) secreted by hybridoma cell lines. The BALBIC mice were immunized several times with PR.RSV-resp strain replicating on porcine alveolar macrophage (PAM), then the spleen cells of the hyperimmunized mouse and SP2IO cells were fused with PEG-1500. The cell fusion rate of 70.6% and positive rate of 6.3% were obtained and two hybridoma cell lines secreting monoclonal antibodies were developed after two subclones. It indicated that the two monoclonal antibodies reacting specifically with 19KD (M) and 45-5OKD (GP3) proteins ofPRRSV by Western-blotting would certainly be useful for research and diagnostic purposes.