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目的原核表达、纯化人星状病毒(human astrovirus,HAstV)非结构蛋白nsP1a/3,并制备多克隆抗体。方法酶切质粒nsP1a/3-pCDNA3.1,获得nsP1a/3基因,克隆至原核表达载体pET-28a,构建重组原核表达质粒nsP1a/3-pET-28a,转化大肠埃希菌BL2(DE3),IPTG诱导表达,并优化表达条件。表达的重组蛋白经Ni2+亲和层析纯化后,经腹腔免疫SD大鼠,共4次,最后1次免疫7 d后,经心脏采血,分离血清,ELISA法检测多抗效价,Western blot鉴定多抗的特异性。结果重组表达质粒nsP1a/3-pET-28a经双酶切和测序鉴定构建正确;表达的重组蛋白相对分子质量约为22500,以包涵体形式表达;纯化的重组蛋白纯度为86.6%,浓度为1.26mg/ml;制备的多克隆抗体效价较高,可达1∶30 000,且特异性较好。结论成功地原核表达、纯化了HAstV非结构蛋白nsP1a/3,并制备了特异性良好的鼠多克隆抗体,为进一步研究nsP1a/3蛋白在病毒侵染靶细胞时的作用机制奠定了基础。
Objective To purify human nonstructural protein nsP1a / 3 from human astrovirus (HAstV) and prepare polyclonal antibody. Methods The nsP1a / 3 gene was digested with restriction enzyme nsP1a / 3-pCDNA3.1 and cloned into prokaryotic expression vector pET-28a to construct recombinant prokaryotic expression plasmid nsP1a / 3-pET-28a. The recombinant plasmid was transformed into Escherichia coli BL2 IPTG induces expression and optimizes expression conditions. After purified by Ni2 + affinity chromatography, SD rats were immunized intraperitoneally for 4 times. After the last immunization for 7 days, the blood was collected from the heart and the serum was separated. The multi-resistance titer was detected by ELISA and Western blot Multi-anti-specific. Results The constructed recombinant plasmid nsP1a / 3-pET-28a was correctly constructed by double enzyme digestion and sequencing. The relative molecular weight of recombinant protein was about 22,500, which was expressed as inclusion body. The purified recombinant protein had a purity of 86.6% and a concentration of 1.26 mg / ml. The prepared polyclonal antibody has higher titer of 1:30 000 and better specificity. Conclusion The prokaryotic expression system of nsst1 / 3, which is a non-structural protein of HAstV, was successfully purified and polyclonal antibody was prepared. This study laid the foundation of further study on the mechanism of action of nsP1a / 3 protein in virus target cells.