| 摘要: |
| 【目的】制备猪流行性腹泻病毒（PEDV）CH-HeB14毒株S1蛋白的多克隆抗体，为 PEDV 新流行毒株诊断试剂盒和疫苗研制提供基础材料。【方法】利用RT-PCR扩增PEDV CH-HeB14毒株S1基因，对序列特征进行生物信息学分析，用PCR将S1基因截成不同片段（S1A、S1B、S1C、S1C-1和S1C-1），分别克隆至原核表达载体pET28a,转化BL21(DE3)构建重组表达菌株；用IPTG诱导重组蛋白表达，并进行SDS PAGE和Western blotting鉴定。将表达蛋白与佐剂乳化制备免疫原免疫兔多克隆抗体，Western blotting和免疫过氧化物酶单层细胞染色法（IPMA）检测抗体的免疫活性。【结果】PEDV S1、S1A、S1B、S1C、S1C-1和S1C-2片段长度依次为2 079，600，600，878，549 和447 bp。系统进化树表明，CH-HeB14株与2010年以来的22个新流行毒株亲缘关系较近，而与经典疫苗株CV777、attenuated DR13亲缘关系较远。成功截短表达了S1蛋白不同区段S1A、S1B和S1C-1重组蛋白(rS1A、rS1B和rS1C-1)，且蛋白均以包涵体形式表达。制备了抗rS1A、rS1B和rS1C-1蛋白的多克隆抗体，其与这3种蛋白反应良好，且抗rS1A重组蛋白的多克隆抗体Western blotting效价为1∶32 000；IPMA结果显示，该抗体能与PEDV CH-HeB14毒株和经典疫苗毒株 attenuated DR13均发生特异性反应。【结论】成功克隆、表达了变异毒株PEDV CH-HeB14的S1基因，所制备的S1蛋白多克隆抗体具有良好的免疫活性。 |
| 关键词: 猪流行性腹泻病毒 S1蛋白 多克隆抗体 |
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| 基金项目:国家自然科学基金青年基金项目（31101837）；衡阳师范学院引进人才专项（16D20）；湖南省教育厅优秀青年项目(17B041) |
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| Expression of porcine epidemic diarrhea virus CH-HeB14 strain S1 protein and preparation of its polyclonal antibody |
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TANG Qinghai,LI Xiaorong,YANG Hai,et al
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| Abstract: |
| 【Objective】This study prepared polyclonal antibody of S1 protein of porcine epidemic diarrhea virus (PEDV) CH-HeB14 strain to provide basis for the development of diagnostic kit and vaccine.【Method】The S1 gene of PEDV CH HeB14 strain was amplified by reverse transcription polymerase chain reaction (RT-PCR),analyzed by bioinformatics software,truncated into different segments (S1A,S1B,S1C,S1C-1 and S1C-1) by PCR,sub-cloned into prokaryotic expression vector pET28a and transformed into BL21(DE3) to obtain the expression recombinants.IPTG was used to induce the expression of recombinant protein before being analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.The expressed proteins were purified and emulsified with immune adjuvant,and the immunogen was inoculated into rabbits to prepare polyclonal antibodies (pAb) against PEDV S1 protein.The immune competence of the pAb was evaluated by immune peroxidase monolayer assay (IPMA) and Western blotting.【Result】The molecular weights of S1,S1A,S1B,S1C,S1C-1 and S1C-2 were 2 079,600,600,878,549 and 447 bp,respectively.Phylogenetic tree showed that PEDV CH-HeB14 strain S1 gene had close relationship with the 22 novel prevailing strains since 2010,while they differed genetically from the vaccine strains (CV777 and attenuated DR13).The truncated proteins (rS1A,rS1B and rS1C-1) were successfully expressed in form of inclusion body (IB).The pAb against these three truncated proteins (rS1A,rS1B and rS1C-1) showed good activity and the Western blotting titer of anti recombinant rS1A protein pAb was 1∶32 000.IPMA results revealed that the pAb showed specific activity when reacting with the attenuated DR13 strain or CH HeB14 strain viruses infected cells.【Conclusion】PEDV CH-HeB14 strain S1 gene was successfully cloned and expressed,and the prepared anti-S1 pAb showed good immune-activity. |
| Key words: porcine epidemic diarrhea virus S1 protein polyclonal antibody |
