欧洲鳗鲡RIG-I基因的克隆、序列特征分析与原核表达

Cloning,sequence characterization analysis and prokaryotic expression of Anguilla anguilla RIG-I gene

为研究欧洲鳗鲡(Anguilla anguilla)RIG-I基因的特性,通过设计引物,扩增其序列,克隆至pCE2-TA/Blunt-Zero载体,测序验证后对所获得欧洲鳗鲡RIG-I基因序列进行生物信息学分析及原核表达。结果表明,欧洲鳗鲡RIG-I基因ORF序列有2823 bp碱基,编码940个氨基酸,与日本鳗鲡(A.japonica)的序列同源性为96.71%;RIG-I蛋白属酸性亲水性蛋白,不存在跨膜结构,无信号肽,较不稳定,主要分布于细胞质中,有6个保守功能区;SDS-PAGE和western blot结果显示,实现了RIG-I基因在大肠杆菌中的高效表达,为后期制备RIG-I多克隆抗体和进一步探索RIG-I介导的天然免疫在鳗鲡抵御鳗鲡疱疹病毒(Anguillid herpesvirus,AngHV)侵染过程中的作用机制提供了研究基础。

Retinoic acid-inducible gene I(RIG-I)is one of RIG-I like receptors(RLRs),a member of DExD/H Box RNA helicase family.There are two cascades of caspase enrichment domains(CARD)at its N-terminal,which are cytoplasmic sensors of viral RNA.They can recognize viral mRNA and activate RLRs to bind to specific ligands during viral invasion,inducing the production of interferon(IFN)and inflammatory factors to resist viral invasion.RIG-I is inclined to recognize short dsRNA and 5′PPP dsRNA,and keeps self-inhibition state without stimulation by dsRNA.RIG-I can recognize both RNA virus and DNA virus,thus RIG-I and its mediated immune response play an important role in the host antiviral immune response during the infection of DNA viruses.Compared with mammals,the acquired immune system of fish is not fully differentiated,and its resistance to pathogen invasion is more dependent on its natural immune system.A large number of studies have shown that the natural immunity mediated by RIG-I plays an important role in the process of fish resisting virus invasion during virus infection.RIG-I is activated and specifically mediates the production of type II IFN-I of zebrafish during the infection by neuronecrosis virus(NNV);RIG-I and regulatory factors in its mediated signaling pathway will be activated in silver crucian carp after carp herpesvirus type II(cyprinid herpesvirus 2,CyHV-2)infection.Previous studies have shown that RIG-I gene and several genes in the RLRs signaling pathway of eel skin mucus are activated after infection by Anguillid herpesvirus(AngHV).In order to study the characteristics of RIG-I gene of Anguilla anguilla,primers were designed to amplify its sequence.Then the target fragment was cloned into pCE2-TA/Blunt Zero vector and verified by sequencing.The sequence characteristics of A.anguilla RIG-I were analyzed.NCBI BLAST online tool was used to analyze the homology of the amplified sequences.The composition and physicochemical properties of nucleotide and amino acid sequences were analyzed by Protparam.Protein transmembrane domain was predicted by TMHMM2.0 analysis.The protein signal peptide structure was predicted by SignALP-5.0.PSORT II was used for subcellular localization prediction.Protein secondary structure was predicted by HNN.The conserved functional domain of gene protein was analyzed by CDD.The ORF sequence fragment of A.anguilla RIG-I gene was cloned into vector pET-30 a and transformed into Escherichia coli BL21(DE3),and then induced by IPTG at 15℃for 16 h for prokaryotic expression.Bacterial bodies were collected by centrifugation,and the cell lysate,lysate supernatant and lysate precipitation were taken for SDS-PAGE analysis.The induced lysate supernatant was subjected to western blot validation of RIG-I expression,with mouse anti-His-Tag monoclonal antibody as primary antibody,and HRP-labeled sheep anti-mouse IgG as secondary antibody.Bioinformatics analysis showed that the ORF sequence of A.anguilla RIG-I gene was 2823 bp and encoded 940 amino acids.The sequence homology of RIG-I gene between A.anguilla and A.japonica was 96.71%.RIG-I might be an acidic hydrophilic protein with no transmembrane structure,no signal peptide and instability,mainly distributed in the cytoplasm,and had 6 conserved functional regions.The results of SDS-PAGE showed that the expected protein bands appeared at the size of about 108 kDa for the cell lysate and precipitation after 16 h of 15℃induction.RIG-I protein in cell lysate precipitation was significantly higher than the supernatant,which indicated that the protein was less soluble and mainly distributed in the inclusion body.The western blot analysis showed that inducible bacteria containing the prokaryotic expression plasmid 30 a-RIG-I were specifically detected at the size of 108 kDa by the His-Tag monoclonal antibody,consistent with the expected size of the expressed protein,whereas the band for the control was about 40 kDa,indicating the realization of A.anguilla RIG-I gene expression in E.coli BL21(DE3).Two RIG-I gene subtypes of A.japonica,AjRIG-I and AjRIG-Ib,were reported.Their homology was only 39%.The RIG-I gene of A.anguilla in this research had 96.71%homology with AjRIG-Ib,and only a few of bases had point mutations,but low homology with AjRIG-I.These results provided reference for the study of A.anguilla RIG-I gene.The bioinformatics analysis showed that RIG-I protein was mainly located in the cytoplasm and relatively unstable,which was consistent with the previously report that RIG-I was a cytosolic sensor of viral RNA and was self-inhibition without stimulation by dsRNA.Mammalian RIG-I proteins generally had four conserved functional regions,from the N-terminal to the C-terminal:two tandem CARD domains,DEx D/H-box RNA helicase domain,and C-terminal domain(CTD).According to the CDD prediction result,RIG-I protein of A.anguilla had six conserved functional regions.The first two belonged to DD superfamily,and the third and fifth belonged to DEAD-like_helicase_N superfamily and DEAD-like_helicase_C superfamily,the fourth and sixth belonged to an alternavtive RIG-I_Family C.DD superfamily was the largest class of protein interaction modules,with a key role in cell apoptosis,necrosis and immune cell signaling pathways.It included four subfamilies:DD,death effector domain(DED),card and pyrin domain(PYD).The two domains,CARD_RIG-I_r1(cd08816)and CARD_RIG-I_r2(cd08817),belonged to the CARD subfamily of DD superfamily,indicating that the RIG-I protein of A.anguilla was similar to that of mammalian RIG-I protein,and there were also two tandem CARDs at the N-terminus.The C-terminal domain of A.anguilla RIG-I protein belonged to RIG-I_C family,i.e.CTD domain.The domain between the DEAD-like_helicase_N-terminal domain and the DEAD-like_helicase_C-terminal domain,belonged to the RIG-I_C family which was different from the C-terminal CTD domain.This structure had not been reported in previous studies on RIG-I protein,and its effect on the function of RIG-I protein in A.anguilla needed to be further verified.The prokaryotic expression of RIG-I protein in A.anguilla provided fundamental research on the preparation of RIG-I polyclonal antibody and the mechanism of RIG-I mediated natural immunity in the process of eel resistance to AngHV infection.