猪伪狂犬病病毒TK/gE/gI缺失株的构建及与Montanide Gel 01佐剂联合免疫效果评价

Construction of porcine pseudorabies virus with TK/gE/gI gene deletion and evaluation of its immune efficacy in combination with Montanide Gel 01 adjuvant

自2011年以来,伪狂犬病病毒(PRV)变异株在我国免疫猪群中大面积流行,对养猪业造成了严重经济损失,且在我国广泛使用的Bartha K-61疫苗已不能为猪群提供完全有效的保护。因此,本研究以rPRV-AH-gE-/gI-为亲本毒株,通过同源重组对TK基因进行缺失,构建了rPRV-AH-TK-/gE-/gI-三基因缺失重组病毒,并对其生物学特性进行了评价。将该病毒单独或与Montanide Gel 01(MG01)佐剂联合免疫小鼠,通过血清中和抗体试验和攻毒保护试验评价了其对小鼠的免疫保护效果。结果显示,重组病毒rPRV-AH-TK-/gE-/gI-构建成功,且有良好的遗传稳定性和生长性能。动物试验结果表明,重组病毒rPRV-AH-TK-/gE-/gI-与佐剂MG01联合免疫效果更好,其中106 TCID50 rPRV-AH-TK-/gE-/gI-+MG01组与106 TCID50 rPRV-AH-TK-/gE-/gI-组相比,小鼠血清中和抗体水平显著升高(P<0.001);在用106 TCID50 PRV-AH攻毒时,无论免疫剂量为106 TCID50或105 TCID50,rPRV-AH-TK-/gE-/gI-与MG01联合免疫均可为小鼠提供100%的保护力。

Since 2011,the pseudorabies virus(PRV)variant strain has been pandemic among immunized pigs in China,causing serious economic losses to the pig industry,and the Bartha K-61 vaccine which was widely used in China cannot provide complete protection for pigs.Therefore,it is necessary to develop new vaccines against the emerging PRV strain to improve the resistance of pigs to PRV.In this study,the TK gene of rPRV-AH-gE-/gI-was deleted by homologous recombination,yielding recombinant virus rPRV-AH-TK-/gE-/gI-,then the biological characteristics of rPRV-AH-TK-/gE-/gI-was examined.The immune efficacy of rPRV-AH-TK-/gE-/gI-alone or in combination with Montanide Gel 01(MG01)adjuvant was evaluated in mice by serum neutralizing antibody assay and challenge test.The results showed that the recombinant virus rPRV-AH-TK-/gE-/gI-was constructed successfully and had good genetic stability and growth characteristics.Animal experiments showed that rPRV-AH-TK-/gE-/gI-in combination with MG01 adjuvant had better immune efficacy.Compared with 106 TCID50 rPRV-AH-TK-/gE-/gI-group,the level of neutralizing antibody of mice in 106 TCID50 rPRV-AH-TK-/gE-/gI-+MG01 group was significantly higher(P<0.001).The immunization dose of 106 TCID50 rPRV-AH-TK-/gE-/gI-or 105 TCID50 rPRV-AH-TK-/gE-/gI-in combination with MG01 could provide protection rate of 100%for mice challenged with 106 TCID50 PRV-AH.