【摘要】 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。更多还原

【Abstract】 Human Cytomegalovirus(HCMV) is ubiquitous in humans, and could cause a variety of diseases. It presents a high infection rate worldwide, which poses a serious threat to the health of human beings. Here,main coding sequence containing neutralizing antigenic epitopes AD-1, AD-2 and AD-3(named AD13) on the HCMV g B was synthesized according to the gene sequence of HCMV AD169. Expression vector p ET-32 a was digested by Eco R I/Xho I, and the coding sequence of neutralizing antigenic epitopes was ligated to p ET-32 a vector to construct the recombinant prokaryotic expression plasmid. Western-blot and indirect ELISA were used to investigate the recombinant expression, purity and immunoreactivity of AD13 protein.The BALB/c mice were immunized with the purified protein, and then the monoclonal antibodies(mAbs) a-gainst AD13 protein were prepared and screened. SDS-PAGE showed that the relative molecular mass of recombinant AD13 protein was 35 kD, and Western-blot and indirect ELISA results revealed that the recombinant AD13 protein, as an antigen, had significant immunoreactivity. After cell fusion and screening,five hybridoma cell strains secreting monoclonal antibodies against AD13 were obtained, which have specificity in reaction with the AD13 antigen. This provides diagnostic raw materials and research foundation for developing HCMV subunit vaccines.更多还原