Prokaryotic Expression Of The H Protein Gene Of Canine Distemper Virus And The Establishment Of Indirect ELISA

Canine distemper virus(CDV) is the pathogen of canine distemper. It’s one of the most serious infectious disease threat dogs healthy, with high mortality rate after onset, but there is no specific treatment. Therefore, this experiment on the basis of CDV N gene, established a canine distemper virus RT-PCR detection method. cloned CDV-H gene, build a prokaryotic expression system and induced H protein expression. Meanwhile,we established a indirect ELISA, and confirmed that the H protein with good immunogenicity. Specific content as follows:1 Primers were designed according to the sequences of CDV gene that retrieved from GenBank, we amplified a gene fragment about 464 bp from CDV nucleic acid.products were analyzed by sequencing,results showed the homology was 94.8%~98.5%.By optimizing the experimental conditions, finally established a canine distemper virus RT-PCR detection method. In the test of 30 clinical samples, using RT-PCR method, the positive detection rate was 90%, higher detection rate and more sensitivity than the detection rate 86.7% that using colloidal gold strip.2 The Hemagglutinin protein can induce strong neutralizing antibodies, thus a set of primers specific to H gene of CDV was designed, which the H protein gene fragment is 1236 nt. Connect the gene fragments with pET32 a, constructed the prokaryotic expression plasmid pET32a-CDVH.Recombinant protein ware efficient expression in Rosetta bacteria. By dialysis method,we obtained recombinant protein H(0.561 mg/m L). The mice immune experiment showed that serum antibody titer of recombinant protein H was higher than 1:10 2400.3 This study used 7.5 ug/m L renaturation of recombinant proteins H Package the enzyme label plate, indirect ELISA was established.The optimum working conditions were determined: the concentration of coating antigen was 7.5ug/ml, coated at 4℃ overnight,blocking with 1% gelatin solution, 80-fold dilution of sera, 1:5000 diluted HRP-antibody, and OPD substrate solution was incubated at 37℃ for 15 min in dark room.The cut off value of the positive results was 0.100. The indirect ELISA method specificity is strong and with good repeatability, Test results of the blood sample which background is clear conform to the expectation.