| Peste des petits ruminants (PPR) is an acute highly contagious and economically important viral disease of small ruminants, especially goats and sheep, with morbidity and mortality rates as high as 100% and 90%, respectively. Peste des petits ruminants is popular in the world also a serious threat to the sheep industry of China.It is important to carry out prevention and control system of etiology, epidemiology,'detection and vaccines on PPR disease in China to promote sheep industry.According to different strains of Peste des petits ruminants virus gene sequences, a pair of primers of N gene sequences and another pair of sheepβ-actin gene were designed, the amplification by the PCR assay was 687bp and 493bp, respectively.RT-PCR assay was developed with the primers for detection of PPR N gene. The detection limit of samples reached 12TCID50/mL in the optimized conditions.Blood samples of sheeps infected with Peste des petits ruminants virus Nigeria 75/1 tested by the RT-PCR was available.Nigeria 75/1 N gene was amplified and cloned to pGEX-4T-1 vector, and induced with IPTG to express the recombinant N proteins, the optimized condition was the LB medium, 37℃,0.2mM IPTG inducing 4h. the recombinant protein was Purified and determinated concentration, immuned BALB/c mice.The B cells was collected from spleen and amalgamated with sp2/0.Six hybridomaclones were selected which can secret positive antibodies. Determination the biological of monoclonal antibody,5 McAb were selected, which were resistansed to head, acid and base.One mAb was selected to develop the cELISA assay and the method was optimized, found that the diluted concentration of antibody was 1:5×104, related antigen was 1:100, the serum between 2-8μL was suitable, blocking buffer 1% BSA was stabily, the effective reaction time was 0.5-2h.Conpetitive with commercial PPR cELISA kit, detection of 176 serum samples, the relative sensitivity of 84%, relative specificity of 97.7%. |
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