论文部分内容阅读
目的:用原核细胞表达柯萨奇病毒B组3型VP1蛋白并观察其免疫效果。方法:构建原核表达质粒pET-his/VP1,转化大肠杆菌BL21(DE3)pLysS,诱导VP1蛋白的表达并纯化。BALB/c小鼠随机分为实验组和对照组。实验组腹腔注射VP1蛋白,对照组注射PBS,每3周免疫1次,共免疫3次。每次免疫后2周取血清,用微量中和试验法检测血清CVB3特异性中和抗体滴度;末次免疫后3周,每组随机取3只小鼠,用细胞计数试剂盒检测淋巴细胞增殖活性和特异性CTL杀伤活性;每组另随机抽取3只腹腔注射3LD50CVB3,第7天检测血中病毒滴度;用致死量的CVB3攻击每组剩余12只小鼠,观察免疫保护作用。结果:实验组小鼠血清中和抗体滴度随免疫次数增加而提高(P<0.05),第3次免疫后中和抗体滴度达70.79±1.31,明显高于PBS对照组(P<0.05);非特异性淋巴细胞增殖活性亦明显高于PBS组(P<0.05),但特异性淋巴细胞增殖活性和CTL杀伤活性两组间无统计学差别(P>0.05);CVB3攻击后,实验组小鼠血中病毒滴度显著降低,生存率达33.33%,而PBS组小鼠无存活,差异有统计学意义(P<0.05)。结论:VP1蛋白可显著增强小鼠的体液免疫应答水平,提高致死量CVB3感染后小鼠的生存率。
Objective: To express VP1 protein of coxsackievirus B group 3 using prokaryotic cells and observe its immune effect. Methods: The prokaryotic expression plasmid pET-his / VP1 was constructed and transformed into E.coli BL21 (DE3) pLysS to induce the expression and purification of VP1 protein. BALB / c mice were randomly divided into experimental group and control group. The experimental group intraperitoneal injection of VP1 protein, the control group injected with PBS, immunized once every 3 weeks, a total of 3 immunizations. Serum was collected 2 weeks after each immunization and the titer of CVB3-specific neutralizing antibody was measured by the method of micro-neutralization test. Three weeks after the last immunization, 3 mice in each group were randomly selected, and the cell counting kit was used to detect the proliferation of lymphocytes 3 CTLs were injected intraperitoneally in each group. 3LD50CVB3 was injected intraperitoneally in each group. The virus titer in blood was detected on the 7th day. The remaining 12 mice were challenged with the lethal dose of CVB3 to observe the protective effect. Results: The titer of serum neutralizing antibody in experimental group increased with the increase of immunization frequency (P <0.05). After the third immunization, the titer of neutralizing antibody reached 70.79 ± 1.31, which was significantly higher than that of PBS control group (P <0.05) ; The proliferation activity of nonspecific lymphocytes was also significantly higher than that of PBS group (P <0.05), but there was no significant difference between the two groups (P> 0.05). But after CVB3 challenge, the experimental group was smaller The virus titer in murine blood was significantly reduced, with a survival rate of 33.33%. There was no significant difference in survival between PBS group and PBS group (P <0.05). Conclusion: VP1 protein can significantly enhance the humoral immune response in mice and increase the survival rate of mice after the lethal dose of CVB3 infection.