伪狂犬病病毒变异株UL43和UL56基因失活株的构建与生物学特性分析

Construction of UL43 and UL56 Gene Inactivation Mutants of Pseudorabies Virus Variant and Analysis of Their Biological Characteristics

为探究UL43与UL56基因失活对伪狂犬病病毒(PRV)变异株毒力的影响,本研究基于伪狂犬病病毒(PRV)变异株AH02LA细菌人工染色体(BAC)BACPRV-G,应用En Passant操作技术,分别将UL43与UL56基因的起始密码子进行点突变,获得两株重组BAC株:BACPRV-G-UL43^(mut)与BACPRV-G-UL56^(mut)。之后将携带同源臂的PRV AH02LA gE/gI基因分别于BACPRV-G-UL43^(mut)和BACPRV-G-UL56^(mut)共转染猪睾丸(ST)细胞,从而获得重组病毒PRV-UL43^(mut)和PRV-UL56^(mut)。生长动力学试验显示PRV-UL43^(mut)病毒滴度在感染后12 h有升高趋势,表明UL43基因可能在感染细胞早期抑制病毒增殖;PRV-UL56^(mut)与亲本毒株PRV AH02LA生长动力学相似,表明UL56基因对病毒生长性能无显著影响。荧光定量PCR试验发现UL56基因可抑制感染细胞早期主要组织相容性复合物Ⅰ类分子的转录。此外,小鼠致病力试验显示,相较于亲本毒PRV AH02LA(LD_(50)=10-3.26/0.2 mL),PRV-UL43^(mut)(LD_(50)=10-2.63/0.2 mL)与PRV-UL56^(mut)(LD_(50)=10-2.19/0.2 mL)对小鼠的致病力均降低,表明UL43基因与UL56基因可能介导病毒对小鼠的致病性。本研究成功构建了PRV变异株UL43、UL56基因失活株,为进一步揭示PRV免疫逃逸的机制奠定基础,也为研制更加安全有效的PRV弱毒疫苗提供理论依据。

To explore the effect of UL43 or UL56 gene inactivation on the virulence of pseudorabies virus(PRV) variant, En Passant technology was used to perform point mutations in the start codons of the UL43 or UL56 genes of the BACPRV-Gto obtain BACPRV-G-UL43mutand BACPRV-G-UL56^(mut). Then co-transfection of DNAs of BACPRV-G-UL43mutand BACPRV-G-UL56^(mut) with PCR fragments of gE/gI gene sequence and homologous sequence of PRV AH02LA into swine testis(ST) cells constructed the recombinant viruses(PRVUL43^(mut)and PRV-UL56^(mut)). Growth kinetics experiments showed that titer of PRV-UL43muton ST cells had an increased trend compared to the parent strain PRV AH02LA at 12-hour post infection, suggesting that UL43gene might inhibit viral proliferation at the early stage of PRV infection. However, the growth kinetics of PRV-UL56^(mut) strain was similar to that of the PRV AH02LA strain, indicating that UL56 gene had no effect on virus growth performance. qRT-PCR experiments found that UL56 gene inhibited the transcription of the major histocompatibility complex class I molecules in infected cells at the early stage of infection. In addition, the pathogenicity test in Balb/c mice showed that PRV-(LD_(50)=10^(-2.63)/0.2 mL) and PRV-UL56^(mut)(LD_(50)=10-2.19/0.2 mL) had a lower pathogenicity in mice compared with the parental virus PRV AH02LA(LD_(50)=10-3.26/0.2 mL), indicating that UL43 and UL56 genes might be involved in mediating the pathogenic effect of PRV on mice. In conclusion, UL43 and UL56 gene inactivation mutants of pseudorabies virus variant were successfully constructed in this study, which laid the foundation for further revealing the escape immunity mechanism of PRV, and also provided a theoretical basis for the development of a safe and effective attenuated PRV vaccine.