猪丁型冠状病毒N蛋白原核表达及其多克隆抗体的制备  

Prokaryotic Expression of Porcine Deltacoronavirus Nucleocapsid Protein and Preparation of Its Polyclonal Antibody

作　　者：刘铭 张永宁[1] LIU Ming;ZHANG Yongning(College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)

机构地区：[1]中国农业大学动物医学院,北京100193

出　　处：《中国畜牧兽医》2022年第7期2698-2707,共10页China Animal Husbandry & Veterinary Medicine

基　　金：财政部和农业农村部:国家现代农业产业技术体系(CARS-35)。

摘　　要：【目的】表达与纯化猪丁型冠状病毒(Porcine deltacoronavirus,PDCoV)的核衣壳(nucleocapsid,N)蛋白,并制备其多克隆抗体(polyclonal antibody,PcAb)。【方法】以PDCoV CHN-HN-1601分离株基因组RNA为模板,利用RT-PCR扩增PDCoV全长的N基因编码序列,将其克隆至原核表达载体pET-28a(+)中构建重组质粒pET-28a-PDCoV-N。经酶切和测序鉴定后,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,利用0.5 mmol/L IPTG于37℃诱导表达12 h。在非变性条件下,利用Ni-NTA琼脂糖树脂从菌体裂解液上清中纯化N-端和C-端均携带6×His标签的重组N蛋白,并将其免疫新西兰白兔制备抗血清。利用Protein A/G琼脂糖树脂从免疫兔的抗血清中亲和层析纯化多克隆抗体,并对多克隆抗体进行Western blotting和间接免疫荧光试验(IFA)鉴定及抗体效价的间接ELISA测定。【结果】重组PDCoV-N蛋白以可溶性和包涵体两种形式表达,分子质量约为42 ku;上清可溶性N蛋白的纯化纯度可达90%、蛋白浓度为0.45 mg/mL;纯化后的多克隆抗体纯度较高,ELISA方法检测其效价为1∶6400,能特异性地识别重组N蛋白和PDCoV,与可引起猪腹泻的猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(PRoV)无交叉反应。【结论】本研究成功制备重组PDCoV-N蛋白及其多克隆抗体,为后续深入研究N蛋白的功能、PDCoV优化血清学检测方法、免疫层析试纸条的制备及开展PDCoV相关基础研究提供参考。【Objective】This study was aimed to express and purify the nucleocapsid(N)protein of Porcine deltacoronavirus(PDCoV)and then prepare its polyclonal antibody(PcAb).【Method】The full-length coding sequence of N gene of PDCoV was amplified by RT-PCR using the genomic RNA of the PDCoV CHN-HN-1601 strain as the template.The resulting amplicon was cloned into the prokaryotic expression vector pET-28a(+)to construct a recombinant plasmid pET-28a-PDCoV-N.After verification by enzyme digestion and sequencing,the recombinant plasmid was transformed into E.coli BL21(DE3)competent cells,which were induced by 0.5 mmol/L IPTG at 37℃for 12 h.The recombinant PDCoV-N protein which carried both an N-terminal and a C-terminal 6×His tag was purified from the supernatant of the lysate of pET-28a-PDCoV-N-transformed E.coli BL21(DE3)using Ni-NTA agarose under native conditions.The purified N protein was used to immunize New Zealand White rabbits to prepare antisera,from which the PcAb was purified by Protein A/G agarose affinity chromatography.The purified PcAb was identified by Western blotting and indirect immunofluorescence assays,and the titer of PcAb was determined by indirect enzyme-linked-immunosorbent assay(ELISA).【Result】The results showed that the recombinant N protein was expressed in both soluble and inclusion body forms with a molecular weight of about 42 ku.The purity of the purified recombinant N protein reached 90%and the protein concentration was 0.45 mg/mL.The purified polyclonal antibody has high purity,the titer of the purified PcAb was 1∶6400,and that the PcAb was able to specifically recognize the recombinant N protein and PDCoV,and had no cross-reaction with other important porcine enteric coronaviruses causing diarrhea in pigs,such as Porcine epidemic diarrhea virus(PEDV),porcine Transmissible gastroenteritis virus(TGEV)and Porcine rotavirus(PRoV).【Conclusion】The successful preparation of recombinant PDCoV-N protein and its PcAb provided valuable biomaterials for the further study of the funct

关 键 词：猪丁型冠状病毒(PDCoV) 核衣壳(N)蛋白 原核表达 多克隆抗体(PcAb) 

分 类 号：S852.651[农业科学—基础兽医学]