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目的:研究人轮状病毒ZTR-5株灭活疫苗的制备及在实验小鼠中的免疫原性评价。方法:轮状病毒ZTR-5株在MA104细胞上经蚀斑筛选纯化后,获得单一克隆接种至Vero细胞上适应性培养,免疫荧光定量检测病毒的感染性滴度,对收获的病毒液进行离心、超滤、分子筛纯化,甲醛灭活,抗原定量检测Al(OH)3吸附制备的实验性疫苗。使用不同剂量(8EU、32EU、128EU、256EU)经肌内注射免疫小鼠,共免疫三次,免疫间隔2周。采用间接ELISA法检测血清特异性抗体效价。结果:通过蚀斑纯化,筛选得到一株纯化的病毒株ZTR-5纯-1,在Vero细胞上适应性后感染性滴度达7.35logCCID50/ml;大量培养收获的病毒原液滴度为7.57logCCID50/ml,制备获得轮状病毒样品抗原含量为2 560EU/ml;经肌内注射,初次免疫后,所有剂量组动物均获得抗体阳转,阳转率为100%;第一次加强免疫后,各组血清特异性抗体水平均明显增高,免疫剂量为128EU和256EU的两组小鼠血清抗体效价均达1∶10 240;第二次加强免疫后,各剂量组(8EU、32EU、128EU、256EU)血清抗体效价依次达1∶5 120,1∶7 456,1∶14 481.54,1∶14 481.54。结论:人轮状病毒ZTR-5株可在Vero细胞上稳定增殖,所制备的疫苗具良好免疫原性,用128EU/2次免疫即可获得良好的免疫效果。
Objective: To study the preparation of human rotavirus ZTR-5 strain inactivated vaccine and evaluate its immunogenicity in experimental mice. Methods: The rotavirus ZTR-5 strain was purified by plaque screening on MA104 cells and single clones were inoculated into Vero cells for adaptive culture. The infectious titer of the virus was detected by immunofluorescence and the harvested virus liquid was centrifuged , Ultrafiltration, molecular sieve purification, formaldehyde inactivation, quantitative detection of antigen Al (OH) 3 prepared by the experimental vaccine. Mice were immunized intramuscularly with different doses (8EU, 32EU, 128EU, 256EU) for three co-immunization intervals of 2 weeks. Serum-specific antibody titers were measured by indirect ELISA. Results: A purified strain of ZTR-5 purified by plaque purification was obtained. The infectivity titer reached 7.35logCCID50 / ml in Vero cells. The titer of virulent virulent strain was 7.57logCCID50 / ml, preparation of rotavirus sample antigen content of 2 560EU / ml; after intramuscular injection, the initial immunization, all doses of animals were positive for the antibody positive, positive conversion rate was 100%; after the first booster immunization, The serum antibody titer of each group was significantly higher than that of the control group (P <0.05). The serum antibody titers of both groups were both 1:10 240 at 128EU and 256EU. After the second booster immunization, the antibody titer of 8EU, 32EU, 128EU, 256EU) serum antibody titers in turn up to 1: 5 120,1: 7 456,1:14 481.54,1: 14 481.54. Conclusion: The human rotavirus ZTR-5 strain can stably proliferate on Vero cells. The prepared vaccine has good immunogenicity, and the immunization effect can be obtained with 128EU / 2 immunization.