表达猪瘟E2基因的重组伪狂犬病毒构建及其 生物学特性分析

Construction of Recombinant Pseudorabies Virus (PRV) Expressing E2 Gene of Classical Swine Fever Virus (CSFV) and Analysis of Its Biological Characteristics

关键词:  伪狂犬病毒  猪瘟病毒  基因缺失  同源重组  生物学特性

Key words:  Pseudorabies virus (PRV)  Classical swine fever virus (CSFV)  Gene deletion  Homologous recombination  Biological characteristics

摘要:

以构建的TK、gI、gE、US9、部分gN基因缺失的绿色荧光标记猪伪狂犬病毒rPRV-BE为亲本株，通过同源重组构建了表达猪瘟病毒(CSFV C株)主要免疫原性基因E2 的重组伪狂犬病毒rPRV-CSFV PE2SC。经PCR、间接免疫荧光试验(IFA)鉴定证实构建正确，并能成功表达E2蛋白。同时，对重组病毒在PK15细胞中的遗传稳定性、增殖特性等进行了研究，结果显示第5、10、15、20代重组病毒经PCR均能扩增出与原代重组病毒相同大小的约为3469 bp含猪瘟病毒E2基因的重组片段；第20代重组病毒感染细胞孔中出现明显的针对E2蛋白的荧光，表明重组病毒在体外连续传20代后仍能稳定遗传。一步生长曲线测定结果表明，与野毒PRV(SX株)、亲本毒rPRV-BE相比，重组病毒rPRV-CSFV PE2SC前期增殖速度较快，病毒滴度到达峰值的时间早，峰值病毒滴度低于野毒，与亲本毒相当，最高病毒滴度依次为106.7TCID50/mL、108.0TCID50/mL、107.0TCID50/mL。结果显示构建的重组病毒具有良好的遗传稳定性及体外复制能力，为进一步对其免疫效果的评价以及PRV基因缺失重组疫苗的研制奠定基础。

Abstract:

Based on one TK、gI、gE、US9 and partial gN gene deletion and green fluorescent labeling strain rPRV-BE, the recombinant pseudorabies virus rPRV-CSFV PE2SC, which expressed the major immunogenicity gene E2 of the classical swine fever virus (CSFV C strain) was constructed by homologous recombination. Through PCR and indirect immunofluorescent assay(IFA), it was proved that the construction was correct and the E2 protein could be expressed successfully. At the same time, the genetic stability and proliferation of rPRV-CSFV PE2SC in PK15 cells were detected. The results showed that the amplified fragment, which containing E2 gene of CSFV, was about 3469bp of the 5th、10th、15th、20th generation recombinant virus by PCR. The length of the amplified fragment is the same as recombinant virus rPRV-CSFV PE2SC amplified with the same primer pairs. What’s more, obvious fluorescence pointed to E2 protein was observed in the 20th generation recombinant virus infected cells, which showed that the recombinant virus has good genetic stability after 20 generations in vitro. One step growth curve showed that compared with wild strain(PRV SX) and parental strain(rPRV-BE), the proliferation of recombinant virus was faster at first and virus titer reached to peak value earlier. The titer of recombinant virus was lower than that of wild virus and equivalent to the parent one. The titer of three virus strains were 106.7TCID50/mL、108.0TCID50/mL、107.0TCID50/mL in turn. The results showed that the recombinant virus rPRV-CSFV PE2SC has good genetic stability and proliferation ability in vitro, which lays a foundation for further evaluation of its immunogenicity and the development of PRV gene deletion recombinant vaccine.