狂犬病毒L蛋白加帽酶功能域的表达、纯化及多克隆抗体的制备与鉴定

Expression,purification,preparation and identification of polyclonal antibodies for the capping enzyme domain of rabies virus L protein

目的克隆狂犬病毒(RABV)L蛋白的加帽酶功能域基因,构建原核表达载体,制备了多克隆抗体,为病毒诊断和抗病毒药物研发提供了有效工具。方法利用基因工程技术,扩增L蛋白加帽酶功能域的基因序列,与双酶切的pET-32a载体连接,获得pET-32a-RABV-L重组表达质粒。经过测序确认无碱基突变,将含pET-32a-RABV-L重组表达质粒的菌液扩大培养后,IPTG诱导表达重组蛋白。通过Ni-NTA亲和层析填料进行纯化,改变不同咪唑洗脱浓度,摸索出适合的洗脱条件。将纯化的蛋白与弗氏佐剂混合免疫小鼠,获得的小鼠多克隆抗体进行间接ELISA检测、Western blot检测、IFA检测。结果成功构建pET-32a-RABV-L原核表达质粒。RABV-L重组蛋白在大肠埃希菌中成功表达,大小约40 ku,且与His-tag的单克隆抗体发生特异性反应。免疫小鼠64 d后,采取的小鼠血清制备多克隆抗体,其工作效价达到25600,在Western blot检测和IFA检测中显示能特异性识别天然病毒L蛋白。结论成功表达、纯化RABV-L蛋白加帽酶功能域的重组蛋白,并成功获得特异性识别天然表位的多克隆抗体。

Objective Cloning of the capping enzyme domain gene of the rabies virus(RABV)L protein,construction of a prokaryotic expression vector,and preparation of polyclonal antibodies have provided effective tools for virus diagnosis and antiviral drug development.Methods The gene sequence of the capping enzyme domain of the L protein was amplified using genetic engineering techniques,ligated with the double-enzyme digested pET-32a vector,resulting in the pET-32a-RABV-L recombinant expression plasmid.After the confirmation of the absence of base mutations by sequencing,the pET-32a-RABV-L recombinant expression bacteria was cultured on a large scale and induced with IPTG to express the recombinant protein.Purification was performed using Ni-NTA affinity chromatography resin,with different imidazole elution concentrations explored to determine suitable elution conditions.The purified protein was mixed with Freund's adjuvant for immunization of mice.The resulting mouse polyclonal antibodies were subjected to indirect ELISA,Western blot,and IFA analyses.Results The pET-32a-RABV-L prokaryotic expression plasmid was successfully constructed.The RABV-L recombinant protein was successfully expressed in Escherichia coli,with a size of approximately 40 ku,and showed specific reactivity with monoclonal antibodies against the His-tag.After 64 days of immunizationof mice,polyclonal antibodies were prepared from mouse sera,with a working titer reaching 25600,demonstrating specific recognition of the natural viral L protein in Western blot and IFA analyses.Conclusion The experiment successfully expressed and purified the recombinant protein containing the capping enzyme domain of RABV L protein,and successfully obtained polyclonal antibodies capable of specifically recognizing natural epitopes.