伪狂犬病病毒gE/gI缺失株CY-6的构建及免疫效力初步研究

Construction and Immogenecity of Pseudorabies Virus CY-6 strain with gE/gI Deletion

构建针对伪狂犬病病毒gE和gI基因缺失的重组转移质粒pBL-gI-gE,将PRV-CY-ΔgI/gE-GFP病毒、pBL-gI-gE转移载体和Lipofectamin 2000转染试剂共转染BHK-21细胞,蚀斑筛选纯化法得到重组病毒PRV-CY-ΔgI/gE,命名为CY-6株。将CY-6株C1代在BHK-21细胞增殖并传至10代,测定生长曲线、病毒含量、基因鉴定、特异性、纯净、毒力和免疫原性。结果CY-6株的生长曲线与亲本CY株无明显变化,C1~C10代毒的病毒含量为10^(8.17)~10^(8.68) TCID50/mL,基因鉴定显示gE基因无366 bp的目的条带,gD基因有1条217 bp的目的条带,gI基因无331 bp的目的条带,特异性检验显示均能被伪狂犬病病毒特异性阳性血清中和;纯净检验该毒种无菌、支原体及外源病毒污染;毒力试验试验猪均5/5健活,免疫原性试验免疫组5/5保护,CY株攻毒对照组5/5发病,其中3/5死亡。以上结果表明伪狂犬病病毒CY-6株符合生产用毒种的初步要求,为研制新型伪狂犬病病毒基因缺失灭活疫苗奠定基础。

BHK-21 cells were co-transfected with porcine pseudorabies virus PRV-CY-ΔgI/gE-GFP,pBL-gI-gE vector and Lipofectamin2000 transfection agent to construct a recombinant virus strain PRV-CY-ΔgI/gE.The CY-6 strain was subject to stain screening and plague purification for further study.Subsequently,BHK-21 cells were inoculated with the CY-6 strain from C1 to C10 passages and the virus harvests were tested for their growth curves,virus contents,gene identification,virulence,purity,specificity and immunogenicity.Results showed that the growth curve of CY-6 Strain had no significant change from its parent CY.The virus contents of C1-C10 passages ranged from 10^(8.17) to 10^(8.68) TCID50/mL.Gene identification showed that the CY-6 had gE and gI genes but possessed gD gene of a 217 bp target band.Purity test demonstrated its sterility for mycoplasma and foreign virus contamination.In addition,the CY-6 was neutralized by specific positive PRV antiserum.In the immunogenicity study,5 pigs of the CY-6 immunized group was all protected and 5 pigs of the challenge control group was infected and 3 pigs died.These results indicated that the CY-6 strain fulfilled the preliminary requirements for the production of virus strains,which laid the foundation for the development of a novel inactivated,gene-deleted PRV vaccine.