• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

猪传染性胃肠炎病毒 S、N基因DNA疫苗载体构建及其免疫原性

Vector Construction and Immunogenicity of S and N Gene DNA Vaccine for TGEV

  • 摘要:
    目的 构建猪传染性胃肠炎病毒(transmissible gastroenteritis virus, TGEV)S、N基因的DNA疫苗载体,并进行免疫原性试验,为猪传染性胃肠炎(transmissible gastroenteritis, TGE)的防控和DNA疫苗研究提供技术支撑和基础数据。
    方法 扩增S基因的A位点、D位点和N基因,并将N基因(单独)、A位点和D位点(融合)克隆至pCDNA3.1-His-C构建重组疫苗载体,运用生物信息学软件预测分析S(A-D)蛋白、N蛋白二级结构组成、三级构像、亚细胞定位和优势B细胞抗原表位。将构建成功的重组载体分别转染至PK-15细胞进行间接免疫荧光试验,运用共聚焦检测重组蛋白的表达分布情况。将重组疫苗载体单独或联合免疫小鼠,运用间接ELISA检测IgG抗体水平。
    结果 扩增出S基因的A位点、D位点和N基因,大小分别为498、606、1149 bp。构建了A位点与D位点(融合)、N基因(单独)的DNA疫苗重组载体p-S(A-D)-His和p-N-His。生物信息学软件预测分析发现TGEV感染宿主细胞时N蛋白主要定位于细胞核和线粒体,S(A-D)蛋白主要定位于细胞质和线粒体,S(A-D)蛋白具有7个优势B细胞抗原表位,N蛋白具有8个优势B细胞抗原表位。重组载体p-S(A-D)-His和p-N-His均在PK-15细胞内成功表达,且S(A-D)-His和N-His在PK-15细胞核和细胞质中均有分布。重组疫苗载体免疫小鼠后,免疫效果由高至低依次为p-N-His>p-S(A-D)-His + p-N-His>p-S(A-D)-His。
    结论 本研究构建了TGEV的 S、N基因的DNA疫苗载体,免疫小鼠后均产生了较强的特异性抗体,为TGEV的核酸疫苗的研制提供了基础材料和依据。

     

    Abstract:
    Objective  DNA vaccine vector of S and N genes of porcine transmissible gastroenteritis virus (TGEV) was constructed with the vaccine immunogenicity detemined to pave the way for studying, preventing, and controling TGE.
    Method  A and D sites on S and N from a TGEV were amplified. The N gene alone as well as the A and D sites fusion were cloned into the vaccine vector pCDNA3.1-His-C. Bioinformatics software was used to predict and analyze the secondary structure, tertiary configuration, subcellular localization, and dominant B cell epitope of S (A-D) and N proteins. The recombinant vectors were transfected into PK-15 cells, and expression distribution of N and the A and D sites fusion detected by indirect immunofluorescence and confocal detection. Mice were immunized with the single or combined recombinant vaccine vector to detect the IgG antibody using indirect ELISA.
    Result  The A and D sites of the S were 498 bp and 606 bp, respectively, and the N, 1149 bp in length. The nucleic acid vaccine expression vectors p-S (A-D)-His and p-N-His for the A and D sites (fusion) and N were constructed. Bioinformatics software predicted that, when TGEV infected the host cells, N protein was mainly located in the nucleus and mitochondria and S (A-D) largely in the cytoplasm and mitochondria, while S (A-D) had 7 and N, 8 dominant B cell epitopes. All p-S (A-D)-His and p-N-His were successfully expressed in PK-15 cells distributed in the nucleus and cytoplasm. The immunized mice showed an effect of immunity in the order of p-N-His>p-S (A-D)-His + p-N-His>p-S (A-D)-His.
    Conclusion The DNA vaccine vectors of S and N of TGEV were successfully constructed. Strong specific antibodies were generated in lab mice after the immunization.

     

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