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目的构建人巨细胞病毒(HCMV)主要被膜磷蛋白pp65(HCMVpp65)基因高表达载体以及VP22.pp65联合表达载体,并评价VP22增强pp65核酸疫苗的体内免疫效果。方法8周龄正常BALB/C雌性小鼠分为pcDNA3.pp65组、pVP22.pp65组、实验对照组,每组7只。利用分子克隆技术构建HCMVpp65表达载体pcDNA3.pp65、VP22与pp65融合基因载体pVP22.pp65,免疫小鼠,四甲基偶氮唑蓝法测定T淋巴细胞增殖反应、白细胞介素(IL)2生物学活性,乳酸脱氢酶释放法测定自然杀伤细胞(NK)活性,酶联免疫吸附实验测定血清HCMVIgM、IgG抗体含量、血清及脾细胞培养液中的IL2、IL4含量。结果小鼠的一般状况始终良好,成功构建了HCMV核酸疫苗载体pcDNA3.pp65、pVP22.pp65;免疫小鼠后,两种疫苗T淋巴细胞增殖反应(8周时刺激指数分别为5.11和5.55)和NK细胞活性(12周分别为8.74%和12.08%)及HCMVIgM(6周时A值分别为1.20和1.58)、IgG抗体(6周时A值分别为1.09和1.78)均高于对照组,且pVP22.pp65组高于pcDNA3.pp65组(P<0.05)。小鼠血清中IL2和IL4含量及IL2的生物学活性较低,各组间差异无统计学意义(均P>0.05);小鼠脾细胞上清中IL2和IL4含量以pVP22.pp65组(411.11pg/ml、76.10pg/ml)最高,IL2生物学活性也以pVP22.pp65组(A值为0.22)最高。结论HCMV核酸疫苗无毒性。pp65
Objective To construct the HCMV high expression vector of pp65 (HCMVpp65) gene and VP22.pp65 expression vector and evaluate the in vivo immune response of VP22 enhanced pp65 nucleic acid vaccine. Methods 8-week-old normal BALB / C female mice were divided into pcDNA3.pp65 group, pVP22.pp65 group, experimental control group, 7 mice in each group. The molecular cloning technique was used to construct HCMVpp65 expression vector pcDNA3.pp65, VP22 and pp65 fusion gene vector pVP22.pp65, immunized mice, T lymphocyte proliferation assay, interleukin (IL) 2 biology NK cell activity was measured by enzyme-linked immunosorbent assay (ELISA). The levels of serum HCMVIgM and IgG were measured by enzyme-linked immunosorbent assay (ELISA). IL-2 and IL-4 levels in serum and splenocyte culture medium were measured. Results The general condition of mice was always good. HCMV DNA vaccine vectors pcDNA3.pp65 and pVP22.pp65 were constructed successfully. After immunized mice, the proliferation of T lymphocytes of both vaccines (stimulation index were 5.11 and 5.55 at 8 weeks) and The NK cell activity (8.74% at 12 weeks and 12.08% at 12.08%, respectively) and HCMVIgM (A values were 1.20 and 1.58 at 6 weeks, respectively) and IgG antibody (A values at 6 weeks were 1.09 and 1.78 respectively) The pVP22.pp65 group was higher than the pcDNA3.pp65 group (P <0.05). The levels of IL2 and IL4 in serum and the biological activities of IL2 in mice serum were lower than those in control group (all P> 0.05). The levels of IL2 and IL4 in the supernatant of the mice spleen cells were detected by pVP22.pp65 group (411.11 pg / ml, 76.10 pg / ml), and IL2 biological activity was also highest in pVP22.pp65 group (A value was 0.22). Conclusion HCMV DNA vaccine is non-toxic. pp65