猪伪狂犬病病毒双基因缺失突变株(HB-98株)安全性、稳定性和免疫原性测定

The Safety,Stablization and Immunogenicity of Double Gene-negative Mutant of Pseudorabies Virus Strain(PrV HB-98)

对以伪狂犬病病毒鄂A株为亲本毒株构建的TK和gG双基因缺失突变株（PrV HB-98株）的增殖能力、安全性、毒力稳定性和免疫原性进行了测定。结果表明，PrV HB-98株在BHK-21细胞上的增殖滴度为10^7.0 TCID50／0．1mL以上，与亲本毒株相当，但高于Bartha株；与PrV鄂A株相比，病毒量为10^7.0TCID50的PrV HB-98株不引起BALB／c小鼠的死亡，毒力也低于Bartha株；将PrV HB-98株在PK-15细胞连续培养25代和在猪体内上连续继代5次，各代次突变株TK基因和LacZ基因能被稳定扩增，未出现毒力回复现象．表明该毒株具有良好的遗传稳定性；以10^5.0、10^6.0、10^7.0TCID50等3个不同剂量的PrV HB-98株接种于妊娠50～60d母猪和1日龄仔猪，母猪均能正常产仔．仔猪也未出现任何临床症状，证明该毒株有较好的安全性。另外，以10^5.0TCID50的PrV HB-98株接种于妊娠50～60d母猪和1日龄仔猪，分别于接种后28d和20d，用10^7.0TCID50 PrV鄂A强毒进行攻击．结果免疫猪都能抵抗强毒的攻击．获得保护，表明该毒株具有很强的免疫原性。综合上述结果表明，PrV HB-98株可以作为候选毒株．用于伪狂犬病基因工程疫苗的研制。

In our previous study, a double gene-negative mutant of pseudorabies virus Ea stain, designated PrV HB-98 strain, was constructed based on the manipulation of TK gene and gG gene of the parent strain. This paper described the measurement of the safety, virulence stabilization and immunogenicity of the mutant. The propagation titers on BHK-21 cells and the virulence to BALB/c mice were measured and compared between PrV HB-98 strain, parental strain and Bartha strain. No death was observed in mice inoculated with 10^7.0TCID50 of the mutant and lower virulence than the other two tested strains was demonstated. The safety test was carried out by inoculating the pregnant sows and 1-day old piglets with 10^5.0,10^6.0 and 10^7.0 TCID50 of the PrV HB-98 strain and the abnormal clinical signs such as reproduce failure in injected sows and illness in inoculated pigets could be found. The stabilization of the virulence and marker gene was evaluated at the cellular and individual level and no virulence reversion was recorded. The mutant was used to immunize the pregnant sows and piglets and these animals were challanged with 100 times dose of the PrV Ea strain at day 28 and 20, respectively, all the immunized pigs received complete protection. The results demonstrated that the mutant possessed highly safety, stable virulence and good immunogenicity, and was suitable to be used as candidate strain for vaccine production.