人巨细胞病毒US31蛋白特异性抗体的制备及鉴定

目的：探讨人巨细胞病毒（HCMV）US31蛋白及其多克隆抗体的制备及鉴定方法。方法：HCMV US31基因经原核密码子优化后进行全基因合成，克隆至pET21a（+）原核表达载体，构建pET21a（+）/HCMV US31重组质粒，测序鉴定；将重组质粒转化大肠杆菌BL21（DE3），IPTG诱导目的蛋白表达，Ni-NTA亲和层析法纯化US31蛋白，免疫新西兰兔制备多克隆抗体，ELISA、Western blot及免疫荧光方法检测抗体效价和特异性。结果：在大肠杆菌中诱导出高表达的HCMV US31融合蛋白，经Ni-NTA亲和纯化后获得高纯度的目的蛋白；免疫新西兰兔诱导产生特异性的IgG抗体，效价为1:49 600；经ELISA、Western blot以及免疫荧光均证实制备的特异性抗体可识别细胞中表达的US31蛋白。结论：成功制备了HCMV US31蛋白及特异性抗体，为进一步研究US31蛋白的生物学功能奠定了基础。

Preparation and identification of human cytomegalovirus US31 protein specific antibody

Objective: To explore the preparation and identification of human cytomegalovirus (HCMV) US31 protein and its polyclonal antibody. Methods: After HCMV US31 gene was optimized by prokaryotic codon for whole gene synthesis and cloned to pET21a (+) prokaryotic expression vector, pET21a (+)/HCMV US31 recombinant plasmid was constructed, with identified sequence. Recombinant plasmid was transformed into Escherichia coli BL21 (DE3), target protein expression induced by IPTG, and US31 protein purified by Ni-NTA affinity chromatography. Immunized New Zealand rabbits were used to prepare polyclonal antibodies, and antibody titer and specificity was detected by ELISA, Western blot and immunofluorescence method. Results: High expression of HCMV US31 fusion protein was induced in Escherichia coli, and high purity target protein was obtained by nickel column affinity purification, and the specific IgG antibody was induced by immune New Zealand rabbits, with the valence being 1:49 600. ELISA, Western blot and immunofluorescence have confirmed that the prepared specific antibodies were able to identify US31 proteins expressed in cells. Conclusion: HCMV US31 protein and specific antibodies were successfully prepared, which laid foundations for further study of the biological function of US31 protein.