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利用基因组中含肌肉特异性miRNA互补序列的修饰型CVB3病毒感染小鼠,研究修饰型病毒在小鼠体内的复制状况以及诱导小鼠免疫保护作用的能力。前期我们在CVB3m株病毒基因组中插入miRNA-133和miRNA-206的互补序列获得修饰型CVB3病毒,命名为CVB-3*T。以对照组(CVB-CON)和紫外灭活野生型CVB3m株病毒组(UV-CVB3-WT)作为对照,测定各组病毒对小鼠的LD50剂量、CVB-3*T对小鼠的致死率、感染小鼠心脏和胰腺的病毒滴度以及心脏病理损伤情况,并检测小鼠血清中炎性细胞因子及中和抗体表达水平以及免疫后小鼠抗病毒能力。结果显示,CVB-3*T对小鼠的LD50明显高于对照组;以相同剂量病毒感染小鼠,CVB-3*T可以明显改善小鼠的生存率,心肌部位的病毒量较对照组明显降低,心肌处病理损伤减轻,而胰腺部位的病毒滴度无明显改变;CVB-3*T组小鼠血清中IFN-γ和TNF-α的含量较UV-CVB3-WT组明显升高,IL-4含量无差异;免疫后,CVB-3*T可诱发机体产生高效价抗CVB3中和抗体,可提高小鼠抵抗病毒再次感染的能力。修饰型CVB3病毒感染可以降低病毒对心肌的亲嗜性,激发小鼠抗病毒免疫,还可以诱导小鼠产生抵抗病毒再次感染的能力。本研究为探索制备CVB3疫苗新途径提供了实验证据。
Mice were infected with the modified CVB3 virus containing the miRNA complementary sequence in the genome to investigate the replication status of the modified virus in mice and the ability of inducing the mice to be immunoprotective. In the early stage, we inserted the complementary sequences of miRNA-133 and miRNA-206 into the viral genome of CVB3m strain to obtain a modified CVB3 virus named CVB-3 * T. The control group (CVB-CON) and UV-CVB3m strain (UV-CVB3-WT) were used as controls to determine the LD50 dose and the lethal dose of CVB-3 * T , The virus titer in heart and pancreas of infected mice and the pathological damage of heart, and the expression of inflammatory cytokines and neutralizing antibodies in mouse serum and the antiviral ability of mice after immunization were detected. The results showed that the LD50 of CVB-3 * T in mice was significantly higher than that in the control group. CVB-3 * T could significantly improve the survival rate of mice in the same dose of virus, and the amount of virus in myocardium was significantly higher than that in control group Reduced the myocardial damage at the pathological lesion, while the pancreas virus titer did not change significantly; CVB-3 * T group of serum IFN-γ and TNF-α levels were significantly higher than the UV-CVB3-WT group, IL -4 was not different. After immunization, CVB-3 * T could induce the body to produce high titer anti-CVB3 neutralizing antibody, which could improve the ability of mice to resist virus re-infection. Modified CVB3 virus infection can reduce the virus’s tropism of myocardium, stimulate mouse anti-virus immunity, mice can also be induced to resist reinfection again. This study provided experimental evidence for exploring new ways to prepare CVB3 vaccine.