Prokaryotic Expression of BPIV3 NP Protein and Its Immunoenhancing Effect on the BPIV3 Inactivated Vaccine

【Objective】 This study was aimed to verify whether the NP protein of Bovine parainfluenza virus type 3 (BPIV3) could enhance the immune effect of BPIV3 inactivated vaccine【Method】 The antigenicity of the protein encoded by NP gene was analyzed by bioinformatics softwares,and the antigenic region was screened.The truncated NP gene sequence of BPIV3 was amplified by PCR and connected to pET-32a(+) plasmid.Then the high-purity BPIV3 NP protein was obtained by E.coli prokaryotic expression system and Ni affinity chromatography.It was confirmed by Western blotting.BPIV3 was inactivated with 0.3% formaldehyde and mixed with Freund's adjuvant 1:1 to prepare inactivated vaccine.Eight New Zealand White rabbits were randomly divided into four groups with two rabbits in each group,including inactivated vaccine group,NP protein group,inactivated vaccine and NP protein mixed group and control group.Blood samples were collected before and every 7 days after immunization.The levels of specific antibodies and neutralizing antibodies in New Zealand White rabbits of the four groups were measured and compared by indirect ELISA and virus neutralization test.【Result】 DNAStar analysis showed that the average antigen index of amino acid region 193-368 of NP protein was 0.4-1.7,and the hydrophilic index was 0-1.5,which proved that this region had strong antigenicity and hydrophilicity.The NP gene was amplified by PCR and the recombinant expression vector was constructed.Gene sequencing showed that the recombinant expression vector was consistent with the expected results.The results of SDS-PAGE showed that NP protein was highly expressed with a molecular weight of 50 ku and expressed in the form of inclusion body.Western blotting showed that the expressed protein had strong reactivity.The results of ELISA showed that 28 days after immunization,the specific antibody titer of the control group was 0,and the specific antibody titers of inactivated vaccine group,NP protein group and inactivated vaccine and NP protein mixed group reached 1:2¹¹,1:2¹⁷ and 1:2¹⁸,respectively.The results of virus neutralization test showed that 28 days after immunization,the neutralizing antibody titer of the control group was 0,and the neutralizing antibody titers of inactivated vaccine group,NP protein group and inactivated vaccine and NP protein mixed group were 1:2^3.32,1:2^4.48 and 1:2^4.98,respectively.【Conclusion】 BPIV3 NP protein could enhance the immune effect of BPIV3 inactivated vaccine.Adding NP protein to the inactivated vaccine could be used as a new vaccination method of BPIV3 inactivated vaccine.