Expression And Application Of Porcine Rotavirus VP7 Protein In Sf9 Insect Cell

Porcine Rotavirus(Po RV)is one of the important pathogen causing diarrhea in piglets It mainly caused acute diarrhea,vomiting,dehydration in piglets.The porcine rotavirus generally does not cause the morbidity to adult pigs,but may cause the horizontal transmission in adult pigs with the latent infection,continuous expelling virus.If the piglets were co-infected the porcine rotavirus with porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV)and pathogenic E.coli,the infected piglets will be aggravated and lead to high mortality rate,and the serious economic losses were caused.Group A rotavirus(RVA)belongs to a non-enveloped virus,it was comprised of 11 segments of double-stranded(ds)genomic RNA that encode for six structural proteins(VP1-4,VP6-7)and six nonstructural proteins(NSP1-6),respectively.The VP7 and VP4 proteins both induced serotype-specific neutralising antibodies and were important for immune protection and vaccine development.To date,27 G types and 37 P types have been identified in humans and animals.The main G types previously identified in pigs are G3,G4,G5 and G11,associated with common P types,i.e.P[6]and P[7].In this study,the fusion protein rBacmid-PoRV-VP7 was expressed in the baculovirus expression system The fusion protein rBacmid-PoRV-VP7 was used to produce monoclonal antibodies against the fusion protein rBacmid-PoRV-VP7.A PoRV antibody detection method with high specificity and sensitivity was established by using fusion protein rbacmid-porv-vp7 as the envelope antigen.1.Construction of recombinant baculovirus expression vector of porcine rotavirus VP7 proteinTo obtain the fusion protein of PoRV-VP7 expressed in baculovirus expression system.In this study,a porcine rotavirus strain GD-01-2015 preserved after laboratory isolation and whole-genome sequencing analysis was used as a template,the vp7 gene was amplified by PCR and cloned into pFast-Bac-HTA vector to construct the transfer vector pFastBacHTA-PoRV-VP7.Then,pFastBacHTA-PoRV-VP7 was transformed into DH10BacTM M competent cells to construct the recombinant baculovirus shuttle plasmid.Next,the rBacmid-PoRV-VP7 recombinant baculovirus was obtained by transfecting into Sf9 insect cells under Lipofectamine TM 3000.The results of the indirect immunofluorescence test(IFA)showed that the fusion protein expressed by the recombinant baculovirus,it can be recognized by the anti-porcine rotavirus antibody and can be expressed in cytoplasm The results of Western-Blot showed that the 37 kDa fusion protein expressed by recombinant baculovirus could be specifically recognized by antibody against His-tagged.2.Preparation of monoclonal antibody against porcine rotavirusIn order to obtain monoclonal antibodies that can be used to establish a rapid detection method for PoRV.In this study,the recombinant baculovirus-expressed protein prepared in chapter 1 was used as an immunogen to immunize 6-8 week-old Balb/c mice.The cell fusion was performed according to conventional methods.Three hybridoma cells which can stable secrete monoclonal antibodies against PoRV-VP7 were screened by indirect immunofluorescence assay(IFA).The three hybridoma cells were named as Mab-PoRV-1G3,Mab-PoRV-2B10,Mab-PoRV-2D2.The results of Western-Blot analysis showed that the three monoclonal antibodies were specific reacted with PoRV.Identification of immunoglobulin the subgrouips of monoclonal antibodies were identificated and the results showed that Mab-PoRV-2B10 belonged to IgG,and the other two strains belonged to IgM.3.Establishment of an ELISA method for detection of pig serum antibodies immunized with porcine rotavirusIn order to establish a rapid detection method for anti-PoRV antibodies with strong specificity and high sensitivity.The recombinant baculovirus-expressed protein prepared in chapter 1 was used as a coating antigen,and HRP conjugated goat anti-swine IgG(H+L)was used as the enzyme-labeled antibody to establish an indirect ELISA method for detection of against PoRV-VP7 antibodies.The specificity test results indicated that the established ELISA method was able to react with the known PoRV-positive pig serum and did not react with known PEDV-positive pig serum and known TGEV-positive pig serum.Sensitivity analysis revealed that the minimum of the ELISA detection method capable of detecting 1:6400 diluted swine serum The results of 92 clinical swine serum samples showed that the coincidence rates of the ELISA test results with the commercial kit test results and the indirect immunofluorescence test results were 76%and 91.3%,respectively.Therefore,the method established in this study can be effectively used to detect anti-PoRV antibodies contained in serum of immunized pigs,and this ELISA detection method will have certain application prospects.