猪流行性腹泻病毒S蛋白原核表达及多克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2022.11.029

Abstract

Abstract: 【Objective】 The purpose of this study was to explore the structure and function of S protein of Porcine epidemic diarrhea virus (PEDV), so as to provide a theoretical basis for promoting the diagnosis of PEDV infection and vaccine development.【Method】 The classic CV777 strain of PEDV was used as the template, the S and the S1 genes fragments were amplified by PCR.The PCR amplification products were cloned into pET-30a (+) prokaryotic expression vector and pFLAG-CMV-3 eukaryotic expression vector respectively, and the prokaryotic expression plasmid pET-30a-PEDV-S and eukaryotic expression plasmid pFLAG-CMV-3-PEDV-S1 were constructed. pET-30a-pEDV-S was transformed into E.coli BL21(DE3) competent cells, and expressed PEDV-S recombinant protein induced by IPTG.The recombinant protein was purified by denaturation and renaturation, and then detected by Western blotting.The recombinant protein PEDV-S was immunized in C57BL mice to prepare polyclonal antibodies.The obtained polyclonal antibody specificity was detected by indirect immunofluorescence assay (IFA) and polyclonal antibody titer was detected by indirect ELISA. pFLAG-CMV-3-PEDV-S1 was transfected into HEK293T cells, and the prepared polyclonal antibody was used as the primary antibody to detect the antigenicity of PEDV-S1 protein and the reactivity of polyclonal antibody by IFA.【Result】 The PEDV S and S1 genes were successfully cloned, and a prokaryotic expression vector capable of expressing recombinant protein PEDV-S and a eukaryotic expression vector capable of efficiently expressing S1 protein in HEK293T cells were constructed.The highest expression of PEDV-S recombinant protein was obtained when IPTG was 0.5 mmol/L and induced at 16 ℃ for 8 h, mainly in the form of inclusion bodies.Western blotting showed that the recombinant protein of PEDV-S was successfully expressed.ELISA results showed that the titer of the polyclonal antibody was 1:3 280 500.IFA results showed that the polyclonal antibody had good specificity and could specifically recognize PEDV-S and PEDV-S1 proteins.【Conclusion】 In this study, PEDV-S recombinant protein and S1 protein were obtained, and polyclonal antibody against PEDV S protein was successfully prepared, which provided conditions for studying the structure and function of S protein and laid a foundation for revealing the pathogenic mechanism of PEDV.

Key words: Porcine epidemic diarrhea virus (PEDV); S protein; protein purification; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65^＋1

ZHANG Tianai, LI Tingting, TAO Xiaoli, LI Tengfei, LIN Jiafeng, HU Siman, LIU Wenxiu, TONG Wei, LI Yonggang. Prokaryotic Expression of Porcine Epidemic Diarrhea Virus S Protein and Preparation and Identification of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4383-4391.

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Prokaryotic Expression of Porcine Epidemic Diarrhea Virus S Protein and Preparation and Identification of Its Polyclonal Antibody

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