论文部分内容阅读
目的表达纯化乙型脑炎病毒非结构蛋白NS5,并制备其多克隆抗体。方法通过PCR法扩增编码NS5蛋白的全长基因,将其克隆到原核表达载体pET28a中,转化大肠杆菌BL21(DE3)进行诱导表达,并通过Ni柱亲和层析纯化重组蛋白。用纯化后的重组蛋白免疫BALB/c鼠制备多克隆抗体。采用间接免疫荧光法检测抗体效价,Western印迹鉴定抗体的特异性。结果与结论成功获得了可溶性表达的NS5蛋白,制备了多克隆抗体,效价为1∶1000,能够特异性识别乙型脑炎病毒感染细胞中表达的NS5蛋白,为进一步研究NS5蛋白的生物学功能奠定了基础。
Objective To express purified non-structural protein NS5 of Japanese encephalitis virus and prepare its polyclonal antibody. Methods The full-length gene encoding NS5 protein was amplified by PCR and cloned into prokaryotic expression vector pET28a. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced to express. The recombinant protein was purified by Ni column affinity chromatography. BALB / c mice were immunized with the purified recombinant protein to prepare polyclonal antibodies. Antibody titers were detected by indirect immunofluorescence and Western blotting was used to identify the specificity of the antibodies. RESULTS AND CONCLUSIONS Soluble NS5 protein was successfully obtained and polyclonal antibody was prepared. The titer was 1: 1000, which can specifically recognize NS5 protein expressed in JE virus infected cells. To further study the biology of NS5 protein Function laid the foundation.