Prokaryotic Expression and Polyclonal Antibody Preparation of ORF3 Protein of Avian hepatitis E virus CaHEV-GDSZ01 Strain

doi:10.7668/hbnxb.20194610

Abstract

Abstract:

The aim of this study was to express the ORF3 protein of Avian hepatitis E virus (aHEV)and prepare its polyclonal antibodies,which can provide biomaterials for further study on the biological function of new vaccine of aHEV ORF3 protein.The ORF3 gene of CaHEV-GDSZ01 strain was amplified by RT-PCR,ligated with pET-32a(+)expression vector to construct recombinant prokaryotic expression plasmid,and transformed into BL21(DE3)competent cells to induce expression.The expression of ORF3 protein was analyzed by SDS-PAGE gel electrophoresis and immunization of rabbits to obtain rabbit anti-ORF3 polyclonal antibody.The titer of polyclonal antibody was determined by indirect ELISA,and its specificity was detected by Western Blot and IFA.The pET-32a-ORF3 recombinant prokaryotic expression plasmid was successfully constructed,and the 27 ku insoluble recombinant protein of aHEV ORF3 was successfully expressed by prokaryotic expression system.The results of Western Blot and IFA showed that the prepared polyclonal antibody could specifically recognize ORF3 protein,and the ELISA results showed that the titer of polyclonal antibody was 1∶51 200.In this study,the ORF3 protein of CaHEV-GDSZ01 strain was successfully expressed and its polyclonal antibody was prepared.

Key words: Avian hepatitis E virus, Polyclonal antibody, Prokaryotic expression, Immunization, ORF3 protein

ZHANG Yuqian, LI Changle, LÜ Zhihang, YAO Xinyan, ZHANG Xuelian. Prokaryotic Expression and Polyclonal Antibody Preparation of ORF3 Protein of Avian hepatitis E virus CaHEV-GDSZ01 Strain[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 224-229. doi: 10.7668/hbnxb.20194610.

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http://www.hbnxb.net/EN/Y2024/V39/I3/224

Figures/Tables 8

Tab.1 Primer sequence information

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	片段预期大小/bp Expected length
F(aHEV-ORF3-Y)	CGCGGATCCATGTGTCTTAGTTGTCAG	263
R(aHEV-ORF3-Y)	CCCAAGCTTCTACATCTGGTACCGTGCGA
F(aHEV-ORF3-Z)	CCGCTCGAGACCATGGGCATGTGTCTTAGTTGTCAG	272
R(aHEV-ORF3-Z)	CGCGGATCCCATCTGGTACCGTGC

Fig.1 Results of PCR amplification of ORF3 gene M.DL2000 Marker;1.Amplification products of aHEV ORF3 gene (prokaryotic);2.Amplification products of aHEV ORF3 gene(eukaryotic).

Fig.2 PCR identification results of recombinant expression plasmid M.DL 2000 DNA Marker;1—8.PCR products of pET-32a-ORF3 bacteria liquid;9—16.PCR products of pCMV-ORF3 bacteria liquid.

Fig.3 Results of SDS-PAGE analysis of ORF3 recombinant protein M.Molecular weight standard of protein;1.Before the induction of pET-32a(+)empty vector;2.After induction of pET-32a(+)empty vector;3.Before the induction of pET-32a-ORF3 vector;4.After induction of pET-32a-ORF3 vector;5.Ultrasonic supernatant;6.Ultrasonic precipitation.

Fig.4 Purification effect of ORF3 recombinant protein (A)and Western Blot identification result(B) M.Molecular weight standard of protein;1.pET-32a(+)empty vector;2.ORF3 recombinant protein.The same as Fig.6.

Fig.5 Detection results of polyclonal antibody titer against ORF3 recombinant protein

Fig.6 Analysis of reactivity and specificity of polyclonal antibodies against ORF3 protein

Fig.7 Indirect immunofluorescence analysis of polyclonal antibodies against ORF3 protein

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