犬冠状病毒N蛋白的原核表达及其多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2020.05.004

摘要/Abstract

摘要： 本研究旨在克隆犬冠状病毒（canine coronavirus，CCV）N基因，体外表达N蛋白，并制备抗该蛋白质的多克隆抗体，用于CCV的诊断及其抗原的检测。参考GenBank中CCV的N基因序列（登录号：KY063618.2），选择CCV流行毒株的N基因，通过对该基因密码子进行优化和基因合成，最后选择一段有效基因构建重组表达质粒pET-B2M-N，将成功构建的重组质粒转化大肠杆菌DH5α感受态细胞，挑取阳性克隆提取质粒，转化大肠杆菌BL21（DE3）感受态细胞，通过0.5 mmol/L IPTG 30 ℃进行诱导表达。结果表明，优化诱导条件后成功表达出大小约为49 ku的重组蛋白。将重组蛋白与弗氏佐剂按一定比例混合，每隔2周免疫G767、G768两只日本大耳白兔数次，用间接ELISA检测G768抗体效价可达1∶512 000，选用G768抗体进行抗体纯化，纯化后浓度可达10 mg/mL，用间接ELISA、Western blotting和间接免疫荧光试验对N蛋白纯化后制备的兔多克隆抗体进行检测分析，表明表达的重组N蛋白免疫原性良好，制备的多克隆抗体具有良好的反应原性。本研究为犬冠状病毒抗原抗体检测以及靶标CCV诊断试剂盒的建立奠定了一定的基础。

关键词: 犬冠状病毒(CCV); N基因; 原核表达; 多克隆抗体

Abstract: The purpose of this study was to clone the N gene of canine coronavirus(CCV),express the N protein in vitro,and prepare a polyclonal antibody against this protein for the diagnosis of CCV and its antigen detection.Referring to the N gene sequence of CCV in GenBank (accession No.:KY063618.2) and the N gene of CCV epidemic strain was selected,and the recombinant expression plasmid pET-B2M-N was constructed by codon optimization and gene synthesis.Finally,an effective gene was selected to construct the recombinant plasmid,which transformed into E.coli DH5α constructer.After sequencing,positive clones were transformed into E.coli BL21 (DE3) strain and induced by IPTG.Finally,IPTG concentration of 0.5 mmol/L was used to induce at 30 ℃.The results showed that the recombinant plasmid pET-B2M-N containing canine coronavirus N gene was successfully constructed.After optimizing the induction conditions,the recombinant protein with a size of about 49 ku was successfully expressed.The recombinant protein was mixed with Freund’s adjuvant to immunize Japanese White rabbits G767 and G768 for several times,and the G768 antibody titer was up to 1:512 000 assayed by indirect ELISA.The antibody could be purified to 10 mg/mL.The charactered of polyclonal antibodies detected by ELISA,Western blotting and immunofluorescence,indicating that the expressed recombinant N protein had good immunogenicity and reactogenicity.This study provides a basis for the detection of canine coronavirus antigen and antibodies and the establishment of target CCV diagnostic kit.

Key words: canine coronavirus(CCV); N gene; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65⁺5

周亚花, 殷娟斌, 刘乐, 殷相平, 李彦敏, 张志东. 犬冠状病毒N蛋白的原核表达及其多克隆抗体的制备[J]. 中国畜牧兽医, 2020, 47(5): 1318-1325.

ZHOU Yahua, YIN Juanbin, LIU Le, YIN Xiangping, LI Yanmin, ZHANG Zhidong. Prokaryotic Expression of Canine Coronavirus N Protein and Preparation of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(5): 1318-1325.

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