Prokaryotic Expression Of G Gene Of Bovine Respiratory Syncytial Virus And Preparation Of Its Polyclonal Antibody

Bovine respiratory syncytial disease is a respiratory disease caused by bovine respiratory syncytial virus（Bovine respiratory syncytial virus,BRSV）.The disease is widely distributed all over the world,and its mortality rate is not high,but its incidence is very high.It usually occurs in winter,causing huge losses to the cattle industry.In order to evaluate the situation of cattle infected with BRSV in Inner Mongolia,302 nose swabs were randomly collected from cattle with mild respiratory symptoms in Bayannur,Tongliao,Wulanhaote,Ordos,Chifeng and Hohhot from March 2018 to May 2019.The positive rate was detected by RT-PCR method and sent to sequence.Then the antigenic region of BRSV G gene was analyzed by biological software,the corresponding nucleotide sequence was found,and a pair of specific primers were designed.Then the BRSV G gene fragment was amplified by RT-PCR and the expression vector PET-32a-G was constructed.Then the expression of G protein was induced and the induction conditions were optimized.The protein was purified by Ni-IDA affinity chromatography,and the protein concentration was determined by BCA protein concentration determination kit.BALB/c mice were immunized with expressed G protein to prepare polyclonal antibody,and the titer of polyclonal antibody was determined by indirect ELISA method,and compared with standard BRSV positive serum.The results showed that the total positive rate of bovine respiratory syncytial virus（BRSV）was 6.95%（21/302）,of which 3.97%was mixed infection of BRSV and BPIV-3.G gene was successfully amplified by using BRSV c DNA as template.The recombinant plasmid p ET-32a-G was constructed correctly.The soluble G recombinant protein with molecular weight of 40 k Da was obtained by induced expression.After optimizing the expression conditions,the concentration of purified and concentrated protein was 2.07 mg/m L.By Western-blot detection,it was found that the protein had reactivity.The polyclonal antibody was successfully prepared,and the highest antibody titer was 2¹⁷.The titer of antibody in standard BRSV positive serum was 2¹⁹.After comparison,it was proved that the titer of polyclonal antibody was good.In summary,this study laid a foundation for the establishment of indirect ELISA detection of BRSV antibody and the development of subunit vaccine.