禽戊型肝炎病毒CaHEV-GDSZ01株ORF2蛋白的生物信息学分析、原核表达及多克隆抗体制备

doi:10.7668/hbnxb.20194919

摘要/Abstract

摘要：

旨在分析禽戊型肝炎病毒(aHEV)CaHEV-GDSZ01株ORF2蛋白的结构和生物学功能等,并利用大肠杆菌原核表达系统表达ORF2重组蛋白及制备抗ORF2蛋白多克隆抗体,验证重组蛋白的免疫原性和反应原性等。利用生物信息学软件对CaHEV-GDSZ01株ORF2蛋白的理化性质、结构和功能等方面进行分析;通过酶切、连接pET-32a(+)载体与CaHEV GDSZ01株的ORF2基因,构建pET-32a-ORF2-His和pET-32a-ORF2原核表达质粒,并转化至DE3感受态细胞中进行诱导表达,分析其表达形式;ORF2重组蛋白经切胶纯化,利用Western Blot试验验证其反应原性后,免疫新西兰大白兔以获得兔抗ORF2蛋白多克隆抗体,并通过Western Blot方法对多抗进行鉴定分析、利用间接ELISA方法进行抗体效价检测。生物信息学分析结果显示,CaHEV-GDSZ01株的ORF2蛋白为亲水性的、不稳定的蛋白,且其存在信号肽的概率高达93.59%,其二级结构主要以无规则卷曲为主,具备结合抗体的结构条件,其保护性抗原整体预测评分则表明ORF2蛋白具备良好的免疫原性。试验成功构建了ORF2重组原核表达质粒,并成功诱导表达了目的蛋白,经SDS-PAGE试验和Western Blot试验显示,诱导表达的蛋白主要为非可溶性蛋白,并具有良好的反应原性;而制备的兔源抗 aHEV ORF2蛋白多克隆抗体的效价为1∶102 400,且能特异性识别免疫原ORF2蛋白、非免疫原ORF2-His蛋白和截短的sORF2蛋白。以上结果初步分析了ORF2蛋白的理化性质和部分生物学功能,成功表达了aHEV ORF2蛋白,制备了抗ORF2蛋白的兔源多克隆抗体。

关键词: 禽戊型肝炎病毒, ORF2蛋白, 生物信息学, 原核表达, 多克隆抗体

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

张玉倩, 吕志航, 刘春红, 廉春杨, 张雪莲. 禽戊型肝炎病毒CaHEV-GDSZ01株ORF2蛋白的生物信息学分析、原核表达及多克隆抗体制备[J]. 华北农学报, 2024, 39(6): 231-238. doi: 10.7668/hbnxb.20194919.

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.

http://www.hbnxb.net/CN/Y2024/V39/I6/231

图/表 12

表1 生物信息学分析软件(服务器)

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

表2 引物序列信息

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

图1 ORF2蛋白亲/疏水性预测

Fig.1 Hydrophilic/hydrophobic prediction of ORF2 protein

图2 ORF2蛋白跨膜区预测(左)和信号肽预测(右)

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

图3 ORF2蛋白二级结构预测 h.α-螺旋;e.延伸链;t.β-转角;c.无规则卷曲。

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

图4 ORF2蛋白三维模型预测

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

图5 ORF2基因PCR扩增结果 M.DL2000 Marker;1.ORF2 PCR产物(引物F1+R);2.ORF2 PCR产物(引物F2+R)。

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).

图6 aHEV ORF2原核表达质粒双酶切鉴定结果 M. DL10000 Marker;1.质粒;2.双酶切产物。

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.

图7 ORF2和sORF2重组蛋白SDS-PAGE分析 M.蛋白分子标准;1.原核载体诱导前;2.诱导后;3.超声破碎上清;4.超声破碎沉淀。

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.

图8 ORF2重组蛋白的纯化效果(A)及Western Blot鉴定(B) M.蛋白分子标准;1.ORF2重组蛋白纯化前;2.ORF2重组蛋白纯化后。

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.

图9 抗ORF2蛋白多克隆抗体反应原性和特异性分析 A.pET-32a-ORF2蛋白;B.pET-32a-ORF2-His蛋白;C.sORF2蛋白。M.蛋白质分子质量标准;1.pET-32a(+)空载体;2.重组蛋白。

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.

图10 抗ORF2重组蛋白多克隆抗体效价检测

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

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