Bioinformatics Analysis,Prokaryotic Expression and Polyclonal Antibody Preparation of ORF2 Protein of Avian hepatitis E virus(CaHEV-GDSZ01)Strain

doi:10.7668/hbnxb.20194919

Abstract

Abstract:

The aim of this study is to analyze the structure and biological function of ORF2 protein of Avian hepatitis E virus (aHEV)CaHEV-GDSZ01 strain,and the recombinant ORF2 protein was expressed in E.coli prokaryotic expression system and polyclonal antibodies against ORF2 protein were prepared to verify the immunogenicity and reactivity of the recombinant protein.The physicochemical properties,structure and function of ORF2 proteins of CaHEV-GDSZ01 strain were analyzed by bioinformatics software.The prokaryotic expression plasmids of pET-32a-ORF2-His and pET-32a-ORF2 were constructed by digesting and ligating pET-32a(+)vector with ORF2 gene of CaHEV GDSZ01 strain,and then transformed into DE3 competent cells for induction and expression,and the expression form was analyzed.The recombinant ORF2 protein was purified by gel cutting,its reactivity was verified by Western Blot test,and then rabbits were immunized to obtain rabbit anti-ORF2 protein polyclonal antibody.The polyclonal antibody was identified and analyzed by Western Blot,and the antibody titer was detected by indirect ELISA.The results of bioinformatics analysis showed that the ORF2 protein of CaHEV-GDSZ01 strain was hydrophilic and unstable,and the probability of the existence of signal peptide was as high as 93.59%.The secondary structure of the protein was mainly irregular curl and had the structural conditions for binding antibodies.And the overall prediction score of protective antigen of ORF2 protein showed that it had good immunogenicity.ORF2 gene prokaryotic expression plasmids was successfully constructed,and the target protein was successfully expressed.SDS-PAGE showed that the protein was expressed as insoluble protein,and the protein was proved to have good reactivity by Western Blot.The titer of the prepared polyclonal antibody was 1∶102 400,and it could specifically recognize immunogen ORF2 protein,non-immunogen ORF2-His protein and truncated expressed sORF2 protein.The physicochemical properties and some biological functions of ORF2 protein was compared and analyzed,the related proteins were expressed successfully,and the rabbit polyclonal antibody against ORF2 protein was prepared.

Key words: Avian hepatitis E virus, ORF2 protein, Bioinformatics, Prokaryotic expression, Polyclonal antibody

ZHANG Yuqian, LÜ Zhihang, LIU Chunhong, LIAN Chunyang, ZHANG Xuelian. Bioinformatics Analysis,Prokaryotic Expression and Polyclonal Antibody Preparation of ORF2 Protein of Avian hepatitis E virus(CaHEV-GDSZ01)Strain[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(6): 231-238. doi: 10.7668/hbnxb.20194919.

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Figures/Tables 12

Tab.1 Bioinformatics analysis software(Server)

软件名称 Software name	网址 Website	预测目的 Prediction purpose
ProtParam	https://web.expasy.org/protparam	理化性质
ProtScale	https://web.expasy.org/protscale	亲/疏水性
VaxiJen v2.0	http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html	保护性抗原
TMHMM Server v 2.0	https://services.healthtech.dtu.dk/services/TMHMM-2.0/	跨膜结构域
Novopro	https://novopro.cn/tools/signalp	信号肽
SOPMA	http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html	二级结构
Phyre 2.0	http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index	三级结构

Tab.2 Primer sequence information

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	片段长度/bp Length
F1(aHEV-ORF2-Y)	CGC GGATCCATGTCGCTGCGTGGATTGC	1 821
F2(aHEV-ORF2-Y)	GGAATTC CATATGTCGCTGCGTGGATTGC
R(aHEV-ORF2-Y)	CCC AAGCTTTCAAGGTGGCGAGGGGAATG

Fig.1 Hydrophilic/hydrophobic prediction of ORF2 protein

Fig.2 The prediction of transmembrane region(left)and signal peptide(right)of ORF2 protein

Fig.3 The prediction of secondary structure of ORF2 protein h.Alpha helix;e.Extension chain;t.Beta turn;c.Random coil.

Fig.4 The prediction model of tertiary structure of ORF2 protein

Fig.5 The PCR amplification of ORF2 gene M.DL2000 Marker;1. PCR products of ORF2(Primer F1+R);2. PCR products of ORF2(Primer F2+R).

Fig.6 The results of identification by double enzyme digestion of prokaryotic expression plasmid aHEV ORF2 M. DL10000 Marker;1.Plasmids;2.Double enzyme digestion product.

Fig.7 SDS-PAGE analysis of ORF2 and sORF2 recombinant proteins M.Molecular weight standard of protein;1.Before the induction of prokaryotic vector;2.After prokaryotic vector induction;3.Ultrasonic crushing supernatant;4.Ultrasonic crushing precipitation.

Fig.8 The purification(A)and Western Blot identification(B)of ORF2 recombinant protein M.Molecular weight standard of protein;1.Before purification of ORF2 protein;2.After purification of ORF2 protein.

Fig.9 Analysis of reactivity and specificity of polyclonal antibodies against ORF2 protein A.pET-32a-ORF2 protein;B.pET-32a-ORF2-His protein;C.sORF2 protein.M.Molecular weight standard of protein;1.PET-32a(+)empty vector;2.Recombinant protein.

Fig.10 The titer detection of polyclonal antibody against ORF2 recombinant protein

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