伪狂犬病毒蛋白激酶基因的PCR扩增及其克隆鉴定

Amplifiction and Cloning of Protein Kinase Gene of Pseudorabies Virus by PCR

以ＢＨＫ－２１细胞单层上增殖的伪狂犬病毒（Ｐｓｅｕｄｏｒａｂｉｅｓｖｉｒｕｓ，ＰＲＶ），经离心浓缩后，用ＳＤＳ－蛋白酶Ｋ消化法分离纯化ＰＲＶ基因组ＤＮＡ。参照ＰＲＶＫａ株和ＮＩＡ－３株蛋白激酶（ＰＫ）基因的ＤＮＡ序列，设计并合成了一对长度为２６ｂｐ和３２ｂｐ的引物，以纯化的ＰＲＶ基因组ＤＮＡ为模板，用ＰＣＲ技术成功地扩增出我国伪狂犬病毒地方株的ＰＫ基因，并将它克隆于ｐＵＣ１９载体。酶切分析结果表明，所获ＰＫ基因克隆在ＰｓｔＩ、ＳｍａＩ、ＸｈｏＩ和ＳａｌＩ上的切点与ＰＲＶＮＩＡ－３株相同。为下一步进行ＰＫ基因的体外缺失和重组，以构建减毒的ＰＫ缺失疫苗株奠定了基础。

seudorabies virus(PRV)was mumplicated on BHK-21 monolayer cells.After concentra-tion with centrifuge,viral DNA was isolated by SD-proteinase K digestion method.According to the sequences of protein kinase genes of PRV Ka and NIA-3 strains,the primers with 26 bpand 32 bp were designed.With pured PRV geneome DNA as template,the PK gene of PRV wasamplified successfully by PCR,and cloned into pUC19 vector.Restriction enzyme analysis showedthat the cIoned PK gene at Smal I,Xho I and Sal I site was the same as that of PRV NIA-3strain。This work laid foundation for constructing the PRV vaccine with deletion of PK gene.