表达多个外源基因的重组伪狂犬病病毒的构建及其细胞培养特性研究  

Generation and characterization of a recombinant pseudorabies virus expressing multiple foreign genes

作　　者：刘燕[1] 田志军[1] 周艳君[1] 仇华吉[1] 童光志[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2007年第2期81-85,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家"863计划"资助项目(2001AA213051)

摘　　要：将SV40启动子控制下的LacZ报告基因表达盒与分别在CMV启动子控制下的含有猪瘟病毒(CSFV)E2基因及猪繁殖与呼吸综合征病毒(PRRSV) GP5基因的表达盒插入到伪狂犬病病毒(PRV)Bartha-K61株TK基因中,通过蓝斑筛选获得了一株插入CSFV E2、PRRSV GP5与LacZ基因的重组伪狂犬病病毒,命名为rPRV-E2-GP5。经Westernblot、间接免疫荧光试验证实E2、GP5基因在重组病毒感染细胞中获得了表达。重组病毒感染Vero、PK-15、IBRS2和CEF细胞后的增殖滴度和致细胞病变特征与亲本病毒比较,无显著差异。本试验结果表明在PRV基因组中可以插入多个外源基因,为进一步研究多价基因工程载体疫苗奠定了基础。In this study, a pseudorabies virus （PRV） transfer vector Pgp5-E2-LacZ that contains a LacZ gene expression cassette, the E2 gene of classical swine fever virus （CSFV） and the GP5 gene of porcine reproductive and respiratory syndrome virus （PRRSV） under the control of human cytomegalovirus （CMV） promoter was constructed. This vector was used to cotransfect with genomic DNA of PRV Bartha-K61 into Vero cells using lipofectamin according to standard transfection procedure. A recombinant PRV harbouring LacZ, E2 and GP5 genes, designated as rPRV-E2-GP5, was generated after ten cycles of blue plague purification and PCR identification. The expression of the E2 and GP5 in rPRV-E2-GP5 infected cells was determined by Western blot and immunofluorescence assay （IFA）. Compared to its parental virus Bartha-K61 strain, rPRV-E2-GP5 showed no obvious difference in virus multiplication and cytopathogenic effects in Vero, PK-15, IBRS2 and CEF cell cultures.

关 键 词：重组伪狂犬病病毒 猪瘟病毒 猪繁殖与呼吸综合征病毒 

分 类 号：Q784[生物学—分子生物学] S852.65[农业科学—基础兽医学]