Gene Sequence Analysis Of IBV Isolates From Some Areas Of Hebei Province And Preliminary Development Of Indirect ELISA

Avian infectious bronchitis is a highly contact,acute respiratory infectious disease in chickens caused by infectious bronchitis virus.The disease spreads quickly and has a high incidence,which has a serious impact on the benefits of the poultry industry.The genome of IBV is prone to mutation and recombination,resulting in a large number of genotypes and serotypes,and weak cross-protection.The genotypes of different regions or the same region may be significantly different.It is important to know the local epidemic strains in time for the prevention and control of the disease.In this study,we carried out related research on the homology and variation analysis of IBV by testing clinical samples.The details are as follows:1.Isolation,identification and sequence analysis of IBV in some areas of Hebei Province: We collected suspected IB samples from some areas of Hebei Province for detection,and sequenced the positive samples.According to the sequence analysis of S1 gene and N gene of 14 positive samples,it was found that 6 strains were similar with LX4 strain,accounting for 43% of the positive samples.At the same time,the homology of S1 gene among 14 isolates was 69.7~99.8%.The results of homology analysis showed that the similarity of N gene was 91.5~99.8%.2.Prokaryotic expression and purification of IBV N protein: The S1 gene and N gene of HB-ZD1909 strain were amplified by designing specific primers,and the recombinant plasmids pgex-6p-1-s1 and pgex-6p-1-n were successfully constructed.The S1 protein and N protein expressed in prokaryotic cells were obtained by IPTG induction.Due to the poor purification effect of S1 Protein,soluble expression and good purification effect of N protein were selected as the basis of follow-up experiments.3.Development of indirect ELISA detection method: An indirect ELISA for detection of IBV was established by using the expressed and purified N protein in prokaryotic cells.After a series of exploration and optimization of conditions,the optimal antigen coating condition of this method is 0.5 ?g/mL,and the most suitable coating condition is 4°C overnight.The negative and positive cutoff value is determined to be 0.0996,the best blocking fluid condition is 5% skim milk,the best blocking time is 2 hours,the serum dilution ratio is 1:100,the best serum action time is 30 min,and the best secondary antibody action time is 60 min,the substrate action time is 10 min.The results of repeatability and specificity test showed that the N protein obtained in this experiment can replace IB virus to detect antibody,which has good application value,and can be used as an important means for rapid diagnosis and immune monitoring.