口蹄疫病毒前导蛋白的原核表达及多克隆抗体的制备

Prokaryotic expression and polyclonal antibody preparation of leader protein of foot-and-mouth disease virus

前导蛋白(Lpro)是口蹄疫病毒(FMDV)编码的重要毒力因子,在拮抗宿主抗病毒反应中发挥多种功能。为了获得抗Lpro的抗体以研究Lpro的功能,本研究将L基因克隆到pET-28a(+)原核表达质粒中,构建了重组表达质粒pET-28a-L,转化至大肠杆菌BL21中进行重组蛋白的诱导表达,所获得重组His-Lpro主要以包涵体形式存在,利用Ni-NTA柱纯化获得了高纯度的His-Lpro,将His-重组Lpro与弗氏佐剂混合乳化后免疫新西兰大白兔,制备了高效价的Lpro兔多克隆抗体血清。通过免疫荧光试验(IFA)、免疫印迹试验(WB)检测Lpro兔多克隆抗体血清的反应原性。结果显示,该多抗血清能够特异性识别FMDV感染BHK21细胞后表达的Lpro,表明本研究制备的Lpro多抗血清具有很好的反应原性,为深入研究Lpro功能及其拮抗宿主天然免疫的作用机理奠定了基础。

Leader protein(Lpro)encoded by foot-and-mouth disease virus(FMDV)is an important virulence factor and plays crucial roles in antagonizing host antiviral response.In order to obtain anti-Lpropolyclonal antibody and study the function of Lpro,the recombinant plasmid p ET-28 a-Lpro was generated by inserting of Lprocoding sequence into prokaryotic expression plasmid p ET-28 a(+),and transferred into E.coli BL21 for expression of recombinant protein.The recombinant His-Lpro was successfully expressed in the form of inclusion bodies,as proved by the reaction of purified His-Lprowith anti-His-tagged monoclonal antibody.Subsequently,New Zealand white rabbits were vaccinated with Freund’s adjuvant-emulsified fusion Lpro,and highly effective rabbit antisera to Lprowere prepared.The result of indirect immunofluorescent assay and Western-blot showed that the prepared rabbit sera prepared.The result of indirect immunofluorescent assay and Western-blot showed that the prepared rabbit sera could specifically recognize Lproexpressed during FMDV infection.The obtained rabbit polyclonal antibody serum to Lprocan be used to further study the function of Lproand its roles in antagonizing host innate immunity.