Preparation and Efficacy of Inactivated Pseudorabies Vaccine Using Different Adjuvants
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摘要:
目的 比较不同种类佐剂制备的伪狂犬病毒（PRV）FJ2012株灭活疫苗的免疫效力。 方法 对PRV新发变异毒株FJ-2012的gE/gI基因采用同源重组技术缺失构建gE/gI基因缺失株（FJ-2012ΔgE/gI株），经甲醛灭活后分别与水相佐剂GEL02、两性佐剂ISA 206VG和白油佐剂制成不同种类的灭活疫苗，经质量检查后免疫绵羊，免疫28 d后采用ELISA法检测各组绵羊的gB和gE抗体，并且以变异毒FJ-2012攻毒，评估不同灭活疫苗对绵羊的保护效力。 结果 成功构建了基因缺失株FJ-2012ΔgE/gI，并制备3种不同佐剂的灭活疫苗，接种绵羊后均无不良反应发生。免疫28 d后gB抗体均全部转阳，gE抗体阴性；对照组绵羊在攻毒后6 d内全部死亡（5/5），以GEL02和白油作为佐剂的灭活疫苗组绵羊全部存活（5/5），保护率达到100%，而以ISA 206VG作为佐剂的免疫组绵羊死亡2头，保护率仅为60%（3/5）。 结论 以GEL02佐剂和白油佐剂制成的FJ-2012ΔgE/gI株灭活疫苗免疫绵羊后可以抵御变异毒亲本株FJ-2012株的攻击，能够对我国目前新发变异PRV的防控提供技术保障。 Abstract:Objective Immune efficacy of inactivated pseudorabies vaccine prepared with different adjuvants were compared. Methods Using 3 different adjuvants as base, inactivated vaccines against the novel variants of pseudorabies virus (PRV), FJ-2012, were prepared from the gE/gI-deleted virus. After a quality assessment, these water-phase GEL02-, amphoteric ISA 206VG- or white oil adjuvant-based vaccines were injected into sheep. Detection of gB and gE antibodies in the animals were performed using ELISA. The protective efficacy against the disease in 28 d after vaccination was determined by comparing to that of an FJ-2012 challenge test. Results The successfully prepared vaccines showed no adverse reactions on the inoculated sheep. The gB antibody was tested positive, but gE negative, in the vaccinated sheep. Out of 5 sheep in the 4 test animal groups, 5 in the challenge control died in 6 d and two perished in the group received ISA 206VG-based vaccine. But all sheep in the group injected with either the experimental GEL02- or the white oil-based vaccine survived. Conclusion A perfect protection rate against FJ-2012 was demonstrated on the sheep vaccinated by the GEL02- or the white oil-based vaccine obtained from the FJ-2012ΔgE/gI strain in this study. A new venue to prevent and control the novel variants of PRV in China was made available. -
Key words:
- Pseudorabies virus /
- gE/gI gene deletion /
- immune efficacy /
- inactivated vaccine
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图 1 转移质粒pUC19:H1:EGFP:BAC:H2转染BHK-21细胞荧光性的鉴定
Figure 1. Green fluorescence of pUC19:H1:EGFP:BAC:H2 plasmid transfected into BHK-21 cell
图 2 pUC19:H1:EGFP:BAC:H2与PRV FJ-2012毒株感染性DNA共转染BHK-21细胞后形成的带有EGFP序列的rPRV:EGFP+:BAC+重组病毒
注：左图为在荧光下的重组病毒，右图为明场下的病毒细胞病变。
Figure 2. Plaques of recombinant PRV rPRV- FJ2012ΔgE/gI:EGFP+:BAC+ from pUC19:H1:EGFP:BAC:H2 plasmid and PRV FJ2012 strain DNA co-transfected into BHK-21 cells
Note: Green fluorescence (left) and CPE (right) are shown in plaques under UV excitation and phase contrast.
图 3 pUC19:H1:H2与rFJ-2012-ΔgE/gI:EGFP+:BAC+毒株的感染性DNA共转染BHK-21细胞后形成的删除EGFP:BAC序列的rPRV FJ-2012-ΔgE/gI重组病毒
注：左图为明场下的病毒细胞病变，右图为在荧光场下的重组病毒（显示无荧光）。
Figure 3. Plaques of recombinant PRV- FJ2012ΔgE/gI from pUC19:H1:H2 plasmid and rPRV- FJ2012ΔgE/gI:EGFP+:BAC+ strain DNA co-transfected into BHK-21 cells
Note: CPE (left) and no-green fluorescence (right) are shown in plaques under phase contrast and UV excitation.
表 1 PRV FJ-2012ΔgE/gI株不同佐剂灭活疫苗免疫绵羊28 d后ELISA测定血清gB和gE抗体结果
Table 1. gB and gE antibodies in sheep vaccinated by different inactivated PRV FJ-2012ΔgE/gI vaccines detected using IDEXX ELISA kit
血清样品
Serum samplegB抗体
PRV gB ELISA*gE抗体
PRV gE ELISA§ISA 206VG佐剂疫苗组 ISA 206VG adjuvant vaccine group 874 0.160（+） 0.961（−） 876 0.151（+） 0.962（−） 877 0.214（+） 0.968（−） 878 0.109（+） 1.032（−） 879 0.165（+） 1.108（−） GEL 02 佐剂疫苗组 GEL 02 adjuvant vaccine group 864 0.206（+） 1.083（−） 865 0.121（+） 0.990（−） 867 0.109（+） 0.966（−） 868 0.114（+） 1.018（−） 869 0.127（+） 1.095（−） 白油佐剂疫苗组 Oil adjuvant vaccine group 870 0.123（+） 1.101（−） 871 0.086（+） 1.050（−） 872 0.149（+） 1.050（−） 873 0.190（+） 1.054（−） 889 0.143（+） 1.009（−） 攻毒阳性对照组 Challenge control group 845 1.038（−） 0.979（−） 846 1.093（−） 1.052（−） 847 1.002（−） 1.051（−） 848 0.976（−） 1.061（−） 849 0.955（−） 1.008（−） 攻毒阴性对照组 Non-challenge control group 859 1.055（−） 1.045（−） 860 1.106（−） 1.021（−） 861 0.972（−） 0.953（−） 862 0.104（−） 1.137（−） 863 0.991（−） 0.984（−） 注：*PRV gB ELISA S/N＜0.60 为gB抗体阳性；PRV gB ELISA 0.70≥ S/N＞0.60 为gB抗体阳性可疑；PRV gB ELISA S/N＞0.70 为 gB抗体阴性；§PRV gE ELISA S/N＜0.60 为gE抗体阳性；PRV gE ELISA 0.70≥ S/N＞0.60 为gE抗体可疑；PRV gE ELISA S/N＞0.70 为gE抗体阴性。
Note: *PRV gB ELISA S/N＜0.60: positive; PRV gB ELISA S/N＞0.60 and≤0.70: suspect; PRV gB ELISA S/N＞0.70: negative. §PRV gE ELISA S/N＜0.60: positive; PRV gE ELISA S/N＞0.60 or ≤0.70: suspect; PRV gE ELISA S/N＞0.70: negative.
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