| ObjectiveThe preparation of porcine epidemic diarrhea virus(PEDV,Porcine epidemic diarrhea virus)the nonstructural proteins NSP1(non-structure proteins protein 1)protein and polyclonal antibody;The interaction factors between PEDV-NSP1 and host protein were revealed.The influence of short hairpin RNA(sh RNA)of NSP1 on PEDV replication was explored,which laied a foundation for further research on PEDV replication mechanism.Methods1.Vero cells were infected with PEDV,and the cell status was observed by optical microscope.After PEDV was amplified,the virus titer detected and calculated by IFA.2.Prokaryotic and eukaryotic expression vectors of NSP1 protein were constructed: PEDV RNA was extracted and c DNA was synthesized by reverse transcription.NSP1 fragment was amplified by PCR.The NSP1 fragment was connected to p GEX-6P-1 vector and p FLAG-CMV-3 vector,and verified by double enzyme digestion and sequencing.3.Purification of PEDV-NSP1 recombinant protein: p GEX-6P-NSP1 was transformed into Escherichia coli BL21(DE3).NSP1 protein was induced by Isopropyl-beta-D-thiogalactopyranoside(IPTG)and the optimal expression conditions were explored.NSP1 protein was purified by GST affinity chromatography.The protein concentration was determined by BCA method,and the expression of NSP1 protein was verified by Western blotting.4.Preparation and verification of PEDV-NSP1 fusion protein polyclonal antibody: The purified PEDV-NSP1 protein was injected subcutaneously as antigen to immunize C57BL/6 mice,and peripheral blood was collected before and after immunization.The specificity,sensitivity and titer of polyclonal antibody were detected by Western blotting and ELISA.After PEDV infected Vero cells,the recognition of polyclonal antibodies in the virus was verified by IFA and Western blotting.After transfection of p FLAG-CMV-NSP1 into 293 T cells,the recognition of the polyclonal antibody in 293 T cells was verified by IFA and Western Blotting.5.Screening host proteins interacting with NSP1 by GST pull-down combined protein spectrometry: Pull down experiment was carried out between recombinant p GEX-6p-NSP1 and Vero cell lysate,and p GEX-6P-1 plasmid protein was used as the control group.After silver staining,the samples were analyzed by mass spectrometry,and the score above 10 was classified as screening criteria.The information and function of proteins were retrieved by logging in Uniprot database.The host protein factors interacting with PEDV-NSP1 were screened.6.Study on PEDV replication by short hairpin RNA(sh RNA)of NSP1: The plasmid PEDV NSP1-sh RNA was successfully constructed by Eco R I/Nco I double digestion and sequencing verification.293 T cells were co-transfected with p FALG-CMV-NSP1 plasmid and PEDV NSP1-sh RNA plasmid,and the inhibition effect of PEDV NSP1-sh RNA was verified by Western Blotting.Vero cells were transfected with Non-Target sh RNA and PEDV NSP1-sh RNA,respectively,and then infected with PEDV.IFA and Western Blotting were used to verify whether PEDV NSP1-sh RNA inhibited PEDV infection.Results1.CPE was observed by optical microscope after PEDV infected Vero cells;PEDV virus was successfully amplified by IFA,and the measured titer was calculated to be 4.0X106 FFU/m L.2.Successful construction of recombinant plasmid NSP1: The prokaryotic expression plasmid p GEX-6P-NSP1 and the eukaryotic expression plasmid p FLAG-CMV-NSP1 were successfully constructed by double enzyme digestion and sequencing.3.The optimal conditions for induction of NSP1 protein were as follows: the concentration of IPTG was 0.8 m M and induction was conducted at 28 ℃ for 12h;The purified protein concentration was 2.8 g/L;The size of NSP1 protein was12 KD.4.Preparation and verification of PEDV-NSP1 fusion protein polyclonal antibody: The purified PEDV-NSP1 protein immunized C57BL/6 mice,and the polyclonal antibody titer was greater than 1:3,2000 by ELISA.Western blotting test showed high specificity and sensitivity.PEDV infected Vero cells were verified by IFA and Western blotting that polyclonal antibodies could recognize PEDV.PFLAG-CMV-NSP1 was transfected into 293 T cells,and it was verified by IFA and Western Blotting that the polyclonal antibody could recognize p FLAG-CMV-NSP1 in 293 T cells.5.Preliminary screening of PEDV-NSP1 interaction host proteins: The samples were screened out as tubulin α chain,nucleolus protein 6,WD repeat domain36,RNA helicase,human epiphyte hydrase,etc.Mitochondrial proteins and other host cell proteins interacting with NSP1 play an important role in the expression and infection function of virus-related proteins in and outside the cell.6.The PEDV NSP1-sh RNA plasmid was successfully constructed by sequencing and agarose gel electrophoresis after double enzyme digestion.Western Blotting detection results and statistical analysis showed that the gray value of bands of PEDV NSP1-sh RNA and p Fl AG-CMV-NSP1 plasmids co-transfected were weaker than those of p Fl AG-CMV-NSP1 plasmids transfected alone.The constructed PEDV NSP1-sh RNA plasmid can inhibit PEDV NSP1.PEDV-infected Vero cells after transfection with Non-Target sh RNA showed fluorescence,and the titer was 4×105 FFU/m L.PEDV infected Vero cells after transfection with PEDV NSP1-sh RNA showed fluorescence,the titer was 4×104 FFU/m L.There was no fluorescence in the group without transfection and infection.ConclusionsThe recombinant plasmid NSP1 was constructed successfully;The recombinant protein NSP1 was purified;Highly sensitive and highly valent polyclonal antibody against PEDV-NSP1 protein was successfully prepared.NSP1 polyclonal antibody can specifically recognize the NSP1 eukaryotic expression plasmid and in PEDV virus infected cells;Proteins interacting with PEDV-NSP1 protein and Vero cells were preliminatively screened;PEDV was successfully amplified;Targeted PEDV-NSP1 sh RNA was successfully constructed,and it could inhibit PEDV replication. |
PDF Full Text Request