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为了研究柯萨奇病毒A组16型(Coxsackievirus A16,CVA16)灭活抗原在小鼠体内所产生免疫保护作用效果,我们选用CVA16临床分离株521-01T,在Vero细胞中进行大量培养,并对培养产物进行甲醛灭活及超速离心纯化。SDS-PAGE和Western blot对纯化的灭活病毒纯度及性质进行初步分析。Al(OH)3+CVA16及单独CVA16抗原,分别经皮下注射免疫雌性ICR小鼠;相同免疫途径、剂量于第14和28d加强免疫2次。ELISA法检测CVA16特异性血清IgG抗体滴度;微量中和试验法鉴定血清中和抗体滴度;酶联免疫斑点试验(ELISPOT)检测特异性T淋巴细胞的活化。对Al(OH)3+CVA16抗原免疫组母鼠所产仔鼠进行脑腔攻毒,检测母传抗体对新生乳鼠的保护作用。结果显示,Al(OH)3+CVA16灭活抗原在小鼠体内能诱生高滴度的特异性抗体,3次免疫后产生的特异性血清IgG抗体滴度最高可达1∶1×105(P=0.000),中和滴度高于1∶256。同时,该抗原还可以诱导特异性T淋巴细胞的活化。以1 000LD50的病毒量脑腔接种48h内新生乳鼠的病毒攻击实验显示,该母传抗体对新生乳鼠具有100%的保护。这一结果表明该灭活CVA16病毒抗原具有较好的免疫原性及保护性,为CVA16灭活疫苗的研究及评价体系提供了参考。
In order to investigate the immunoprotective effect of Coxsackievirus A16 (CVA16) inactivated antigens in mice, we used CVA16 clinical isolate 521-01T to conduct a large number of culture in Vero cells, The products were cultured for formaldehyde inactivation and ultracentrifugation purification. SDS-PAGE and Western blot analysis of purified inactivated virus purity and nature of a preliminary analysis. Al (OH) 3 + CVA16 and CVA16 alone. The female ICR mice were immunized subcutaneously with the same immunization route. The dose was boosted twice at 14 and 28 days. The titer of CVA16-specific serum IgG was detected by ELISA. The serum neutralizing antibody titers were identified by micro-neutralization assay. The activation of specific T lymphocytes was detected by ELISPOT. The protective effects of maternal antibodies on the newborn suckling mice were detected by intracerebroventricular injection of the offspring born to the Al (OH) 3 + CVA16 antigen immunized group. The results showed that the high titer specific antibodies could be induced by Al (OH) 3 + CVA16 inactivated antigens in mice and the specific serum IgG antibody titer after 3 immunizations was up to 1: 1 × 105 P = 0.000), neutralization titer higher than 1: 256. At the same time, the antigen can also induce the activation of specific T lymphocytes. Virus challenge experiments with newborn suckling mice inoculated intracerebroventricularly with a dose of 1000 LD50 for 48 hours showed that the mother antibody had 100% protection for newborn suckling mice. This result indicates that the inactivated CVA16 virus antigen has good immunogenicity and protection, and provides a reference for the research and evaluation system of CVA16 inactivated vaccine.