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目的原核表达呼吸道合胞病毒(RSV)非结构NS1蛋白,并对其免疫特性进行研究。方法采用PCR技术获得NS1基因,将其插入pET41a(+)载体上,导入大肠杆菌后诱导表达;利用GST亲和层析柱对诱导表达的NS1蛋白进行纯化,免疫动物制备抗体,Westernblot法鉴定抗体的特异性。结果重组质粒经酶切鉴定和DNA序列测序完全正确,经IPTG诱导后融合蛋白在大肠杆菌BL21-DE3中得到高效表达,表达产物经超声破碎和GST亲和层析纯化获得重组蛋白,分子量约42000Mr,以抗NS1蛋白的多克隆抗体为一抗进行Westernblot检测,结果只有RSV感染细胞的样品在15000Mr处有特异性带显示,其他病毒感染的细胞和阴性对照没有相应条带。结论 NS1基因得到高效表达,免疫制备得到的抗体与RSV感染细胞有特异性反应,与其他常见呼吸道病毒感染细胞无交叉免疫反应。
Objective To express the unstructured NS1 protein of respiratory syncytial virus (RSV) in prokaryotic cells and study its immunological properties. Methods The NS1 gene was amplified by PCR, inserted into pET41a (+) vector and induced into E. coli. The NS1 protein was purified by GST affinity chromatography and immunized to prepare the antibody. Western blotting was used to identify the antibody Specificity. Results The recombinant plasmid was verified by restriction enzyme digestion and sequencing of DNA sequence. The fusion protein was highly expressed in Escherichia coli BL21-DE3 induced by IPTG. The recombinant protein was purified by GST affinity chromatography and purified by ultrasonic. The molecular weight was about 42000Mr Western blot was used to detect the anti-NS1 protein polyclonal antibody. As a result, only the samples with RSV infected cells showed a specific band at 15000Mr, and the other virus-infected cells did not have the corresponding band with the negative control. Conclusion The NS1 gene is highly expressed. The antibody produced by immunization has specific reaction with RSV infected cells and no cross-immunoreaction with other common respiratory virus infected cells.