In order to construct Senecavirus A expressing Strep-tag Ⅱ,Strep-tag Ⅱ was inserted into the C-terminus of the VP1 gene of SVA-I212V/S460L infective clone by overlapping extended PCR method.The reconstructed infectious clone was then transfected into BHK-21 cells to rescue the recombinant virus,and the supernatant was collected after CPE was detected.BHK-21 cells were infected with the virus supernatant and wild type SVA,respectively,and the BHK-21 cells without virus infection were used as negative control.Cells infected with recombinant virus were identified 24 hours after infection by indirect immunofluorescence assas(IFA),mouse anti-SVA VP2 protein MAb 5D10 and mouse anti-Strep-tag Ⅱ MAb were used as primary antibodies,respectively.IFA results exhibited that both SVA-WT and rSVA-Strep infected cells showed specific fluorescence signals when mouse anti-SVA VP2 protein MAb 5D10 was used as the primary antibody,while only recombinant virus infected cells showed specific fluorescence signals when anti-Strep-tag Ⅱ MAb was used as the primary antibody.None of the negative controls showed a special fluorescent signal,indicating that the Strep-tag Ⅱ-labeled recombinant virus was successfully rescued and named rSVA-Strep.To determine the genetic stability of rSVA-Strep,the recombinant virus was passaged in BHK-21 cells for 10 continuously generations.The recombinant virus gene was amplified with primer VP1-F/VP1-R every 2 generations,and the amplified gene of the 10th generation was sequenced.The result displayed that the target bands of about 780bp were present in all the amplified recombinant virus genes,and no mutantion was detected in Strep-tag Ⅱ gene of the 10th generation,indicating that Strep-tag Ⅱ gene was stable in rSVA-Strep replication.BHK-21 cells were infected with either SVA-WT or rSVA-Strep with a multiplicity of infection(MOI)of 0.01,and the virus were harvested at 4 hours,8 hours,12 hours,24 hours,36 hours,48 hours and 72 hours after infection and the virus titer was determined.The results showed that the growth curve of rSVA-Strep and SVA-WT were basically consistent within 36 hours of viral infection,indicating that the insertion of Strep-tag Ⅱ had no obvious effect on the replication of SVA.rSVA-Strep was fully inactivated by 0.2%formaldehyde,and the inactivated virus was purified by Strep-Tactin® XT Purification kit.The inactivated rSVA-Strep was added to Strep-Tactin affinity chromatographic column,the column was then washed by different volumes of elution buffer to elute the virus.And the purification efficiency was evaluated by western blot.The results showed that VP0 and VP2 bands appeared in E1,E2 and E3,among which E1 and E2 showed the most obvious bands,demonstrating that the optimal elution volume was about 2.2 CV.These results indicate that the Strep-tag Ⅱ on the C-terminal of capsid protein VP1 specifically binds to the Strep-Tactin column,which is conducive to the rapid purification of SVA antigens.In this study,Strep-tag Ⅱ tag was successfully inserted into SVA,which can provide technical means for the rapid purification of SVA inactivated vaccine,and also provide reference for the establishment of rapid purification methods for other pathogens.
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