过表达TPL2蛋白PK-15细胞系的建立及其功能验证

Establishment and preliminary application of a PK-15 cell line overexpressing porcine TPL2 gene

通过基因合成的TPL2(tumor progression locus 2)质粒,获得稳定表达TPL2的PK-15/TPL2细胞系。将慢病毒载体Lv-PCDH和TPL2(Pig)重组慢病毒载体Lv-TPL2(Pig)利用转染试剂将其转染至PK-15细胞。感染48 h后,加入嘌呤霉素进行药物筛选,通过筛选得到的阳性细胞株PK-15/TPL2即为稳定表达TPL2蛋白的PK-15细胞株。通过qRT-PCR法和琼脂糖凝胶电泳、IFA,TCID50技术验证PK-15/TPL2细胞系和对口蹄疫病毒(foot-and-mouth disease virus,FMDV)和猪塞内加谷病毒(seneca valley virus,SVV)病原复制的影响。结果显示,细胞表达大量的TPL2的基因产物,用FMDV、SVV病毒感染稳定表达TPL2的PK-15细胞株时,明显抑制病毒的复制。结果表明,利用该技术可高效快速地建立了稳定高表达TPL2的PK-15细胞系,该细胞株的建立为病毒的分离及TPL2的生物学活性研究奠定理论基础。

A PK-15/TPL2 cell line stably expressing TPL2 was obtained by gene synthesis of TPL2(Tumor Progression Locus 2)plasmid.Lentiviral vectors Lv-PCDH and TPL2(Pig)recombinant lentiviral vectors Lv-TPL2(Pig)were transfected into PK-15 cells using transfection reagent.After 48 hours of infection,puromycin was added for drug screening,and the positive cell line PK-15/TPL2 obtained by screening was a PK-15 cell line overexpressing TPL2 protein.qRT-PCR and agarose gel electrophoresis,IFA technique,TCID50 were used to detect the expression of TPL2 mRNA in stably transfected cells PK-15/TPL2 and PK-15/PCDH cells,and further stably expressed by FMDV and SVV infection.PK-15/TPL2 cell line.The results showed that,using the method of the present invention,the cells expressed a large amount of the TPL2 gene,and significantly inhibited the replication of the virus when the PK-15/TPL2 cell strain stably expressing TPL2 was infected by FMDV and SVV virus.The PK-15 cell line stably expressing TPL2 was established efficiently and rapidly.The establishment of this cell line laid the foundation for the isolation of virus and the biological activity study of TPL2.