Prokaryotic expression and polyclonal antibody preparation of porcine deltacoronavirus N protein
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摘要: 【目的】 构建猪德尔塔冠状病毒（Porcine deltacoronavirus,PDCoV）核衣壳（N）蛋白原核表达系统并制备多克隆抗体，为新型疫苗、诊断试剂研发及PDCoV致病机制的深入研究提供基础。【方法】 提取PDCoV HeN17株RNA，经RT-PCR扩增，将N基因克隆至pGEX-6P-1载体构建重组原核表达质粒，转化大肠杆菌BL21(DE3)感受态细胞并进行IPTG诱导表达。利用Glutathione Sepharose 4B亲和层析纯化重组蛋白，采用3C蛋白酶去除标签。以纯化后的N蛋白为免疫原免疫3月龄新西兰大白兔制备多克隆抗体，采用间接酶联免疫吸附测定（ELISA）法测定抗体效价，并通过Western blotting、间接免疫荧光试验（IFA）和免疫沉淀（IP）试验对其进行鉴定。【结果】 成功构建原核表达载体pGEX-6p-1-PDCoV-N，获得的PDCoV-N-GST重组蛋白主要以可溶性形式表达，大小约69k D，经去除GST标签可获得单一目的蛋白条带，经测定蛋白浓度为12.6 mg/mL，获得的PDCoV-N蛋白能与PDCoV阳性血清反应产生单一特异性条带。制备的兔源抗PDCoV-N多克隆抗体效价达1∶4096000，可特异性识别p CAGGS-Flag-N转染HEK-293T细胞中表达的Flag-N蛋白和PDCoV感染LLC-PK1细胞中表达的PDCoV-N蛋白，且能检测到PDCoV-N真核表达载体pCAGGSFlag-N转染HEK-293T细胞中表达的PDCoV-N蛋白。【结论】 成功构建高效PDCoV-N原核表达系统，获得纯度好、浓度高且免疫原性佳的PDCoV-N蛋白，制备的兔源抗PDCoV-N多克隆抗体效价高，具有较强亲和力和特异性，可用于Western blotting、IFA和IP检测。
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关键词:
- 猪德尔塔冠状病毒(PDCoV) /
- N蛋白 /
- 原核表达 /
- 多克隆抗体
Abstract: 【Objective】 A prokaryotic expression system for the nucleocapsid（N） protein of porcine deltacoronavirus（PDCoV） was constructed, and polyclonal antibodies were prepared to provide reference for the development of novel vaccines, diagnostic reagents, and in-depth research into the pathogenic mechanisms of PDCoV. 【Method】 RNA from the PDCoV-HeN17 strain was extracted and amplified by RT-PCR, and the N gene was cloned into the pGEX-6P-1 prokaryotic expression vector to construct a recombinant prokaryotic expression plasmid. This plasmid was transformed into Escherichia coli BL21（DE3） competent cells, and expression was induced with IPTG. The recombinant protein was purified using Glutathione Sepharose 4B affinity chromatography, and the tag was removed with 3C protease. Polyclonal antibodies were prepared using the purified N protein as the immunogen to immunize 3-month-old New Zealand white rabbits.The antibody titer was determined by indirect enzyme-linked immunosorbent assay（ELISA）, and the antibodies were characterized by Western blotting, indirect immunofluorescence（IFA） and immunoprecipitation（IP） assays. 【Result】 The prokaryotic expression vector pGEX-6p-1-PDCoV-N was successfully constructed, and the obtained PDCoV-N-GST fusion protein was primarily expressed in a soluble form, with an approximate size of 69 kD. After the GST tag was removed, a single band of the target protein was obtained, with a concentration determined to be 12.6 mg/mL. The produced PDCoV-N protein could react with PDCoV positive serum to produce a single specific band. The prepared polyclonal antibody against PDCoV-N from rabbits reached a titer of 1∶4096000 and could specifically recognize the Flag-N protein expressed in HEK 293T cells transfected with pCAGGS-Flag-N and the PDCoV-N protein expressed in LLC-PK1cells infected with PDCoV. It could also detect the PDCoV-N protein expressed in HEK 293T cells transfected with the eukaryotic expression vector pCAGGS-Flag-N. 【Conclusion】 An efficient prokaryotic expression system for PDCoV-N is successfully constructed, yielding PDCoV-N protein with high purity, concentration and good immunogenicity. The prepared anti-PDCoV-N polyclonal antibodies from rabbits exhibited high titers, strong affinity, and specificity, making them suitable for applications in Western blotting, IFA, and IP assays. -
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