人星状病毒非结构蛋白nsP1a／1表达纯化及多克隆抗体的制备

摘要

Objective To express and purify nsP1 a/1, the human astrovirus (HAstV) non⁃structural protein, and to prepare its polyclonal antibodies by immunizing animals. Methods The nsP1 a/1 gene sequence of HAstV was amplified by PCR and inserted into the E. coli prokaryotic ex⁃pression vector. The recombinant protein nsP1 a/1 was purified by the Ni⁃affinity chromatography and evaluated by sodium dodecyl sulfate⁃polyacrylamide gel electrophoresis ( SDS⁃PAGE ) and bicinchoninic acid ( BCA ) assay. Male rats were immunized by the purified protein of nsP1 a/1. ELISA was used to evaluate the antibody titer, and Western blotting to detect the specificity of the antibody. Results The prokaryotic expression vector nsP1 a/1⁃pET28 a was successfully construc⁃ted and transformed to the E. coli BL21 ( DE) to express the recombinant proteins. The titer of poly⁃clonal antibody measured by ELISA was 1: 406 374. Conclusion This study successfully identified the purified non⁃structural protein nsP1 a/1 of HAstV by using the prokaryotic expression vector, which lays a foundation for studies on the clinical diagnosis of the replication and infection of HAstV.%目的：表达纯化人星状体病毒（ human astrovirus， HAstV）非结构蛋白nsP1a／1，免疫动物制备多克隆抗体。方法利用PCR技术扩增nsP1a／1基因序列，构建到大肠埃希菌原核表达系统中表达重组nsP1a／1蛋白，使用镍柱亲和层析法对重组蛋白进行纯化，十二烷基磺酸钠⁃聚丙烯酰胺凝胶电泳（ SDS⁃PAGE）和二噻啉甲酸（BCA）实验对重组蛋白的纯度与浓度进行分析，以重组的nsP1a／1蛋白为抗原，免疫雄性SPF级SD 大鼠获得多抗血清，用 ELISA 测定抗体效价、 Western 印迹检测抗体特异性。结果nsP1a／1⁃pET28a原核表达载体构建成功，将其转化至大肠埃希菌BL21（DE3）细菌中诱导表达了重组蛋白，免疫大鼠获得的多抗血清几何平均效价达到1∶406374。结论本实验成功地运用原核表达系统表达并鉴定了人星状体病毒非结构蛋白nsP1a／1，为进一步研究人星状病毒的复制及病毒感染的临床诊断奠定基础。