To prepare polyclonal antibodies(PAbs)against the spike protein(S)of swine acute diarrhea syndrome coronavirus(SADS-CoV),this study amplified the C-terminal domain of the S1 subunit(S1-CTD)encoding the SADS-CoV S protein(384bp)through PCR.The fragment was cloned into a prokaryotic expression vector pGEX-6p-1 to construct the recombinant plasmid pGEX-6p-1-S1-CTD.After verification by double enzyme digestion and sequencing,the recombinant plasmid was transformed into competent cells of Escherichia coli BL21(DE3).Protein expression was induced with IPTG and the reactogenicity of recombinant S1-CTD protein(rS1-CTD)was confirmed by western blot,which showed a specific band at 40ku.After induction,the rS1-CTD was added with an appropriate amount of urea,resuspended,and centrifuged.After SDS-PAGE detection,the target protein gel was cut and crushed.PBS was added to the rS1-CTD and centrifuged after frozen and thawed repeatedly.The collected supernatant was the purified protein.The concentration of rS1-CTD was 33μg/mL,measured using a BCA protein assay kit.The recombinant protein was emulsified with Freund's adjuvant,and New Zealand white rabbits were immunized thrice,followed by blood collection one week after the third immunization to isolate serum and obtain PAbs against S1-CTD.After SADS-CoV infection of Vero E6 cells for 24 hours,rabbit PAbs were used as the primary antibody in both western blot and indirect immunofluorescence assay(IFA).The western blot revealed a specific band at approximately 250ku but not in the negative control.The IFA showed green fluorescence in the infected cells,with no fluorescence in the negative controls.For immunohistochemical(IHC)analysis,ileal tissues from infected piglets were prepared into pathological sections,with the prepared PAbs as the primary antibodies.The tissue sections displayed a brown positive signal,contrasting with the negative control piglet ileal sections,indicating a specific immunoreactivity with the corresponding antigen in ileal tissue of piglets infected with SADS-CoV.Overall,the prepared PAbs against S(S1-CTD)protein demonstrated good reactogenicity and immunogenicity,suitable for detecting SADS-CoV infection in vitro and in vivo through western blot,IFA,and IHC.This study provides a foundation for further development of detection methods for SADS-CoV and studies on the biological function of the S protein.
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