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人巨细胞病毒(Human Cytomegalovirus,HCMV)分布广泛,能够引发多种疾病,人群感染率极高,严重威胁到人类健康。现根据标准毒AD169基因序列,人工合成包膜糖蛋白B(glycoprotein B,g B)AD13区段的主要中和抗原表位AD-1、AD-2、AD-3,并将其与表达载体pET-32a连接,构建重组表达质粒;用IPTG诱导表达重组蛋白,采用Western-blot进行特异性鉴定;将纯化后的重组蛋白免疫BALB/c小鼠,取免疫小鼠脾细胞和SP2/0细胞融合,然后筛选制备AD13单克隆抗体(monoclonal antibody,m Ab)。12%SDS-PAGE电泳鉴定表明重组蛋白表达成功,其相对分子质量约为35 kD;Western-blot和间接ELISA结果说明,重组AD13蛋白作为抗原具有良好的免疫反应性;克隆化后筛选到5个AD13单克隆抗体细胞株,经间接ELISA鉴定,与AD13抗原有良好的特异性反应。上述研究为研发HCMV快速诊断试剂盒提供了原料,也为研究HCMV亚单位疫苗提供了相关的基础。
Human Cytomegalovirus (HCMV) is widely distributed and can cause various diseases. The human infection rate is extremely high, which seriously threatens human health. AD-1, AD-2 and AD-3, the main neutralizing epitopes of AD13 in glycoprotein B (g B) AD, were synthesized based on the standard AD169 gene sequence. pET-32a. The recombinant protein was induced by IPTG and identified by Western-blot. BALB / c mice were immunized with the purified recombinant protein and immunized spleen cells and SP2 / 0 cells Fusion, and then screening for the preparation of AD13 monoclonal antibody (monoclonal antibody, m Ab). 12% SDS-PAGE electrophoresis showed that the recombinant protein was expressed successfully, and the relative molecular mass was about 35 kD. Western-blot and indirect ELISA showed that the recombinant AD13 protein had good immunoreactivity as antigen. After cloning, 5 AD13 monoclonal antibody cell line, identified by indirect ELISA, and AD13 antigen has a good specific reaction. The above research provides a starting material for the development of the HCMV rapid diagnostic kit and also provides a relevant basis for the study of the HCMV subunit vaccine.