PEDV、TGEV与PDCoV S蛋白表位基因三联疫苗的构建及其鉴定

Construction and Identification of a Triple Vaccine of PEDV,TGEV and PDCoV S Protein Epitope Gene

[目的]构建一种同时预防猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪德尔塔冠状病毒(PDCoV)的三联表位疫苗,以预防上述病原导致的猪的相关疾病。[方法]本研究运用免疫信息学在线软件对3种猪肠道冠状病毒(SeCoVs)的S蛋白B、T细胞表位进行预测分析,构建新的表位肽段,命名为PPT,通过ExPASy、VaxiJen、TMHMM、SOPMA、SOLpro和AlphaFlod2等在线软件对PPT进行生物信息学分析,并利用C-IMMSIM在线软件对其免疫反应进行模拟。通过T2A将PPT与PEDV的COE序列、TGEV的SAD序列及PDCoV的CTD序列连接并构建至真核表达载体pEGFP-N1,经PCR和双酶切鉴定正确后,将获得的重组质粒转染HEK293A细胞,经DAPI染色、CCK8、RT-PCR及Western blotting试验验证重组质粒在体外表达情况。[结果]构建的表位蛋白PPT由17条表位肽段组成,经生物信息学软件分析,该蛋白为非跨膜蛋白,结构稳定,具有抗原性和可溶性,亲水性高,无过敏性。C-IMMSIM结果显示,PPT能引起机体树突状细胞(DC)增加,B、T细胞免疫反应,刺激免疫球蛋白IgG、IgM和细胞因子γ-干扰素(IFN-γ)、白细胞介素-2(IL-2)水平升高。重组质粒经PCR和双酶切鉴定结果显示,分别在3359、2375、1064、944、764和467 bp出现特异性目的条带,与预期相符;重组质粒转染HEK293A细胞后,可见绿色荧光;RT-PCR扩增分别在3359、2375、1064、944、764和467 bp处获得与目的基因大小相符的条带;CCK-8检测结果表明,重组质粒对细胞均无明显毒性作用;Western blotting检测结果显示,分别在31.7、16.1、37.9和27.5 ku处出现与目的蛋白分子质量大小一致的条带,重组质粒成功在HEK293A细胞中表达。[结论]本研究基于计算机软件分析设计的PPT表位蛋白成功构建三联疫苗,且在体外表达,为评价PEDV-TGEV-PDCoV三联表位疫苗的免疫效果提供依据。

[Objective]The aim of this experiment was to construct a triple epitope vaccine against Porcine epidemic diarrhoea virus(PEDV),Porcine transmissible gastroenteritis virus(TGEV)and Porcine delta coronavirus(PDCoV)simultaneously to prevent swine-associated diseases caused by the above pathogens.[Method]In this study,immunoinformatics online softwares were used to predict and analyze the B and T cell epitopes of the S protein of three types of Swine enteric coronavirus(SECoVs).A new epitope peptide named PPT was constructed.PPT was subjected to bioinformatics analysis using online softwares such as ExPASy,VaxiJen,TMHMM,SOPMA,SOLpro and AlphaFold2.The immune response was simulated using the C-IMMSIM online software.PPT was connected to the COE sequence of PEDV,the SAD sequence of TGEV,and the CTD sequence of PDCoV through T2A and cloned into the eukaryotic expression vector pEGFP-N1.After PCR and double enzyme digestion identification,the obtained recombinant plasmid was transfected into HEK293A cells.DAPI staining,CCK8,RT-PCR and Western blotting tests were performed to verify the expression of the recombinant plasmid in vitro.[Result]The constructed epitope protein PPT consisted of 17 epitope peptides.According to the bioinformatics analysis of software,the protein was a non-transmembrane protein with stable structure,antigenicity,solubility,high hydrophilicity,and no allergenicity.The results of C-IMMSIM showed that PPT could increase the number of DC cells in the organism,induce B and T cells immune responses,and stimulate the levels of immunoglobulins IgG,IgM and cytokines interferon-γ(IFN-γ)and interleukin-2(IL-2).PCR and double enzyme digestion identification of the recombinant plasmid showed specific target bands at 3359,2375,1064,944,764 and 467 bp,corresponding to the expected sizes.After the transfection of the recombinant plasmid into HEK293A cells,green fluorescence was observed.RT-PCR amplification yielded bands matching the size of the target gene at 3359,2375,1064,944,764 and 467 bp,respectively.CCK-8 assay results indicated that the recombinant plasmid had no significant cytotoxic effect on cells.Western blotting analysis showed specific bands at 31.7,16.1,37.9 and 27.5 ku,corresponding to the molecular weights of the target genes,confirming the successful expression of the recombinant plasmid in HEK293A cells.[Conclusion]Based on the PPT epitope protein designed by computer software analysis,the triple vaccine was successfully constructed and expressed in vitro,which provided a basis for evaluating the immunological effect of the triple epitope vaccine PEDV-TGEV-PDCoV.