小反刍兽疫病毒F蛋白的原核表达及其多克隆抗体的制备

Prokaryotic expression of Peste des petits ruminants virus F protein and preparation of polyclonal antibody

目的原核表达小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)F蛋白,并制备多克隆抗体。方法根据GenBank中登录的PPRV Nigeria 75/1株F基因全长序列,采用DNAStar软件设计去除F基因信号肽和跨膜区的引物,进行RT-PCR扩增,并克隆至原核表达载体pET-32a(+)中,转化E.coli BL21(DE3),经IPTG诱导表达。表达的重组蛋白纯化后,进行SDS-PAGE和Western blot分析,免疫BALB/c小鼠,制备多克隆抗体,并初步应用于PPRV的检测。结果重组原核表达质粒pET-32a(+)-PPRV-F经双酶切和测序证实构建正确;表达的重组F蛋白相对分子质量约59 000,诱导6 h表达量最高,且主要以包涵体形式表达;纯化的重组蛋白纯度可达95%以上;免疫小鼠制备的多抗能与纯化的重组蛋白发生特异性反应,抗体效价高于1∶64 000;间接免疫荧光试验显示,制备的多抗能够识别PPRV Nigeria 75/1株全病毒抗原。结论原核表达了PPRVF蛋白,并制备了高效价的多克隆抗体,为其进一步研究及小反刍兽疫临床快速检测方法的建立奠定了基础。

Objective To express Peste des petits ruminants virus（PPRV） F protein in prokaryotic cells and prepare its polyclonal antibody.Methods According to the full-length gene sequence of PPRV Nigeria 75 / 1 strain reported in GenBank,the primers without signal peptide and transmembrane domain were designed by using DNAStar software,based on which F gene was amplified by RT-PCR and inserted into prokaryotic expression vector pET-32a（＋）.The constructed recombinant plasmid pET-32a（＋）-PPRV-F was transformed to E.coli BL21（DE3） for expression under induction of IPTG.The expressed protein were purified,then identified by SDS-PAGE and Western blot.Polyclonal antibody was prepared by immunizing BALB / c mice with the purified protein and preliminarily used for the determination of PPRV.Results Both restriction analysis and sequencing proved that recombinant plasmid pET-32a-PPRV-F was constructed correctly.The expressed recombinant F protein,with a relative molecular mass of about 59 000,mainly existed in a form of inclusion body and reached a purity of more than 95% after purification,of which the expression level reached the maximum 6 h after induction.The polyclonal antibody prepared by immunizing the mice showed specific reaction with purified recombinant F protein,of which the titer reached more than 1 ∶ 64 000.Indirect IFA showed that the prepared polyclonal antibody recognized the whole virus antigen of PPRV Nigeria 75 / 1 strain.Conclusion PPRV F protein was expressed in prokaryotic cells,and high titer polyclonal antibody was prepared,which laid a foundation of further study on F protein and rapid determination of PPRV in clinic.