Establishment And Application Of Two Indirect ELISA Methods Based On Prokaryotic Expression Of Recombinant Protein Of Bovine Nebovirus And Bovine Norovirus VP1

Bovine Nebovirus(NeV)and Bovine Norovirus(BNoV)belong to the genus Neb virus and Norovirus of the family Caliciviridae respectively.All of them are new pathogens causing calf diarrhea in China,and they are single-stranded positive-chain RNA virus.NeV-VP1 and BNoV-VP1 are the main capsid proteins of the two viruses,which contain abundant epitopes.Because there is no suitable isolation and culture system,although there have been studies on the preparation of specific antibodies by immunizing animals with virus-like particles expressed in eukaryotic systems or the establishment of ELISA detection methods as coated antigens,there are some problems such as complex antigen preparation process and high cost,and this method has not been widely used.The purpose of this study is to express NeV-VP1 and BNoV-VP1 recombinant proteins through E.coli expression system,study their antigenicity,and apply them to the establishment of indirect ELISA method.The following results have been achieved:1.The complete NeV-VP1 and BNoV-VP1 recombinant proteins were successfully expressed in prokaryotic expression system for the first time.Capsid protein VP1 plays an important role in virus infection and immunity.The purpose of this study is to prepare NeV-VP1 and BNoV-VP1 recombinant proteins with E.coli expression system.The signal peptide,transmembrane region,secondary structure,tertiary structure and linear B cell epitope prediction analysis of NeV-VP1,BNoV-VP1 amino acid typing sequence is completed through online server.Codon preference optimization,sequence synthesis and insertion between Nde I and Xho I endonuclease sites of p ET28a(+)were used to construct p ET28a-VP1-BL21(DE3)expression vector expressing the complete coding frame of VP1.The expression was induced by IPTG and the recombinant protein was verified by Western Blot.The results showed that NeV-VP1 and BNoV-VP1 do not have signal peptides and transmembrane structures.The secondary structure contains more than 60%of the random coil structure.The tertiary structure shows that the random coil structure is clearly exposed and contains no intramolecular disulfide structure.The linear B cell epitope prediction results show that the S region of NeV-VP1 and BNoV-VP1 contains more potential epitopes than the P1 and P2 regions.The sizes of recombinant NeV VP1 and BNoV VP1 genes are 1707 bp and 1629 bp respectively,and the N-terminal carries His tag and connecting sequence.The target protein is expressed in the form of inclusion body,and the size is about 60 KDa,as expected.Western-blot results show that the purified target protein is NeV-VP1 and BNoV-VP1 recombinant protein with a purity of 89%and 95%.The results of this study showed that the prokaryotic expression vector which could efficiently express the recombinant proteins of NeV-VP1 and BNoV-VP1 was successfully constructed,which provides a material basis for the preparation of NeV and BNoV specific antibodies and the establishment of NeV and BNoV serological diagnostic methods.2.The recombinant proteins of NeV-VP1 and BNoV-VP1 expressed in E.coli have good antigenicity.In order to verify the antigenicity of NeV-VP1 and BNoV-VP1 prokaryotic recombinant proteins,bovine serum naturally infected with NeV and BNoV was used as the first antibody,and its reactivity was verified by Western Blot test.Rabbits were immunized with NeV-VP1 and BNoV-VP1 recombinant proteins and 201 adjuvant,and the two recombinant proteins were used as coating antigens to establish indirect ELISA to detect the growth and decline of rabbit antibodies.The results showed that the prokaryotic recombinant proteins of NeV-VP1 and BNoV-VP1 could react specifically with NeV and BNoV positive bovine serum.Both NeV-VP1 and BNoV-VP1 recombinant proteins could stimulate rabbits to produce specific antibodies,which increased with the increase of immunization times.In the NeV experimental group,the value of OD_450nmincreased to 0.857 at 2 weeks after the first immunization,and reached the maximum to 1.572 at the third immunization,and then tended to be stable.In the BNoV experimental group,the value of OD450nm increased to 1.101 at the second week of the first immunization and 2.206 at the third immunity,and then tended to be stable.The results of this study show that the prokaryotic recombinant proteins NeV-VP1 and BNoV-VP1 have good reactivity and immunogenicity and can be used as coating antigens to establish indirect ELISA methods for detection of anti-NeV and BNoV.3.An indirect ELISA method for detection of bovine NeV and BNoV specific antibodies was successfully established.The purpose of this study was to establish an indirect ELISA method for detection of specific antibodies against NeV and BNoV for serological investigation of corresponding viruses.Using NeV-VP1 and BNoV-VP1 recombinant proteins as coating antigens,indirect ELISA was established by optimizing coating conditions and reaction conditions,as well as specificity,sensitivity and repeatability tests.Antibody detection was carried out in 482 yak sera.Results the established indirect ELISA for detection of NeV ELISA and BNoV antibodies could not cross react with their virus antibodies except for their positive sera,and the OD_450nm was lower than the critical values of 0.235 and 0.195.The minimum detection limits for NeV and BNoV positive sera were 1:12000 and 1:24000.The coefficients of variation between and within batches were less than 10%.The positive rate of NeV antibody in healthy yak serum and diarrhea yak serum was 15.38%and 15.00%respectively,and the total positive rate was 15.35%,the positive rate of BNoV antibody was 9.50%and 10.00%respectively,and the total positive rate was 9.54%.The results showed that the indirect ELISA,with high sensitivity,good specificity and low cost has been successfully established in this study,which provides a reliable means for seroepidemiological investigation of NeV and BNoV.It provides a reference for the prevention and treatment of calf diarrhea in some areas of Sichuan Province.