| 摘要: |
| 【目的】构建和制备具有较强抗原性的犬埃立克体（Ehrlichia canis）重组蛋白并研究其免疫学特性。【方法】利用ABCpred等在线软件预测分析犬埃立克体 gp19、gp140和gp200等蛋白的优势线性抗原表位，构建多表位融合抗原基因E.canis-megp19及E.canis-megp-1，经PCR扩增后将其克隆至表达载体pET 32a(+)中，构建原核表达载体pET-32a(+)-E.canis-megp19和pET-32a(+)-E.canis-megp-1，经EcoRⅠ和XhoⅠ双酶切鉴定正确后转化至感受态细胞大肠杆菌 BL21 （DE3）中，优化表达条件（IPTG诱导浓度、温度及时间），表达重组蛋白后检测其免疫原性。【结果】成功构建并合成了多表位融合抗原基因E.canis-megp19和E.canis-megp-1，其PCR产物在1.5% 琼脂糖凝胶电泳后分别出现大小为414和429 bp的片段；重组载体pET-32a(+)-E.canis-megp19和pET-32a(+)E.canis-megp-1双酶切后得到了预期长度的目标片段；SDS-PAGE结果显示，当加入1.0 mmol/L IPTG、37 ℃诱导8 h后,E.canis-megp19及E.canis-megp-1重组蛋白均可大量表达，其分子质量分别为38 和39 ku；Western blot分析显示，该重组蛋白可被犬埃立克体阳性血清特异性识别，证实其具有良好的反应原性；小鼠免疫试验表明，该重组蛋白可诱导机体产生特异性抗体。【结论】制备的多表位融合重组蛋白能够高效地在大肠杆菌中表达且能够诱导机体产生特异性抗体。 |
| 关键词: 犬埃立克体 gp19 gp140 gp200 多表位融合抗原基因 |
| DOI： |
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| 基金项目:国家十三五重点研发计划子课题（2016YFD0501005）；兵团中青年科技创新领军人才计划项目（2016BC001） |
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| Immunological characteristics research of multi-epitope fusion antigen protein of Ehrlichia canis |
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LI Chongyang,MENG Qingling,QIAO Jun,et al
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| Abstract: |
| 【Objective】This study constructed Ehrlichia canis protein with strong immunogenic antigen and studied its immunological characteristics.【Method】The dominant linear antigen epitopes of gp19,gp140 and gp200 proteins were analyzed by ABCpred software,and two multi-epitope fusion antigen genes E.canis-megp19 and E.canis -megp-1 were constructed after codon optimization.Then they were cloned by PCR and subcloned into the expression vector pET-32a(+) to generate pET-32a(+)E.canis-megp19 and pET-32a(+)-E.canis-megp-1,respectively.The recombinant expression plasmids assayed by EcoRⅠ and XhoⅠ double enzyme digestion were transformed into E.coli BL21 (DE3).The recombinant proteins were expressed by IPTG with optimal conditions of IPTG concentration,inducing temperature and time,and the immunogenicity of recombinants protein was analyzed.【Result】Multi-epitope fusion antigen genes E.canis-megp19 and E.canis-megp-1 were constructed and synthesized.PCR products showed 414 and 429 bp fragments on 1.5% agarose gel electrophoresis,respectively.Expected target strip was obtained through the double enzyme digestion of recombinant vectors pET-32a(+)-E.canis-megp19 and pET-32a(+)-E.canis-megp-1 by EcoRⅠ and XhoⅠ.SDS PAGE showed that recombinant proteins E.canis-megp19 and E.canis-megp-1 with molecular weights of 38 and 39 ku were expressed largely 8 h after induction with 1.0 mmol/L IPTG at 37 ℃.Western blot analysis indicated that recombinant proteins could be specifically identified by positive serum of E.canis.The mouse immunity test showed that recombinant protein could induce specific antibody.【Conclusion】The prepared multi epitope fusion recombinant proteins can be efficiently expressed in E.coli and can induce specific antibodies. |
| Key words: Ehrlichia canis gp19 gp140 gp200 multi-epitope fusion antigen gene |
