小反刍兽疫核酸疫苗的制备及免疫效果评估
Preparation and evaluation of immune effect of peste des peties ruminants nucleic acid vaccine
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摘要: 为了制备针对小反刍兽疫病毒(Peste des peties ruminants virus, PPRV)F基因的核酸疫苗,并评价其对小鼠的免疫效果,试验采用DNA重组技术将F基因、优化密码子后的F基因以及连接乙型脑炎病毒信号肽(JEVSP)的优化密码子后的F基因3个目的片段分别克隆到真核表达载体pcDNA3.1(+),构建重组质粒pcDNA3.1(+)-F、pcDNA3.1(+)-F-opt和pcDNA3.1(+)-JEVSP-F-opt,大量培养重组质粒菌后,以碱裂解法将菌体悬浮裂解,经中空纤维切向流超滤和微滤浓缩处理,将制备的核酸疫苗以100μg/只的剂量给Balb/c小鼠肌肉注射免疫,共分为4组,pcDNA3.1(+)-F组、pcDNA3.1(+)-F-opt组、pcDNA3.1(+)-JEVSP-F-opt组和pcDNA3.1(+)空载体组,每隔14 d免疫1次,共免疫3次,每次免疫14 d后利用中和试验检测抗体水平。结果表明:成功制备核酸疫苗pcDNA3.1(+)-F、pcDNA3.1(+)-F-opt和pcDNA3.1(+)-JEVSP-F-opt,免疫Balb/c小鼠14 d后pcDNA3.1(+)空载体免疫组中和抗体阳性率为0,pcDNA3.1(+)-F组、pcDNA3.1(+)-F-opt组、pcDNA3.1(+)-JEVSP-F-opt组抗体转阳性,且pcDNA3.1(+)-F-opt组、pcDNA3.1(+)-JEVSP-F-opt组抗体水平呈逐渐增加的趋势。说明成功制备小反刍兽疫核酸疫苗,并且能在小鼠上产生抗体。Abstract: The aim of the study was to prepare the nucleic acid vaccine of F gene of Peste des peties ruminants virus（PPRV） and evaluate its immune effect on mice. In this study, F gene, F gene after optimized codon, and F gene after optimized codon linking Japanese encephalitis virus signal peptide（JECSP） were cloned into eukaryotic expression vector pcDNA3.1（+） by DNA recombination technique to prepare recombinant plasmid pcDNA3.1（+）-F, pcDNA3.1（+）-F-opt and pcDNA3.1（+）-JEVSP-F-opt. After a large number of recombinant plasmid bacteria were cultured, the bacteria were suspended and lysed by alkali pyrolysis, then concentrated by microfiltration and ultrafiltration of hollow fiber purifier. Balb/c mice were immunized by intramuscular injection of nucleic acid vaccine at a dose of 100 μg/mouse. The mice were divided into four groups: pcDNA3.1（+）-F group, pcDNA3.1（+）-F-opt group, pcDNA3.1（+）-JEVSP-F-opt group and pcDNA3.1（+） empty vector group. Immunization was given once every 14 days for three times. Neutralization test was used to detect antibody level after 14 days of each immunization. The results showed that nucleic acid vaccines pcDNA3.1（+）-F, pcDNA3.1（+）-F-opt and pcDNA3.1（+）-JEVSP-F-opt were successfully prepared. After immunizing Balb/c mice with pcDNA3.1（+） empty vector for 14 days, the positive rate of neutralizing antibody was 0. pcDNA3.1（+）-F, pcDNA3.1（+）-F-opt, and pcDNA3.1（+）-JEVSP-F-opt groups were positive, and the antibody level of pcDNA3.1（+）-F-opt, and pcDNA3.1（+）-JEVSP-F-opt groups showed a trend of gradual increase. The results indicated that PPRV vaccine was successfully prepared and the antibody could be produced on mice.
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