Abstract:

To construct an adenoassociated virus (AAV) vector coexpressing the herpes simplex virus 1 thymidine kinase (HSV1tk) and human interleukin1 receptor antagonist(hIL1Ra) gene by utilizing an internal ribosome entry site(IRES) sequence to link the two cDNAs, and to detect its expression in synovial cells. MethodsThe HSV1tk、 IRES and hIL1Ra were amplified by the polymerase chain reaction(PCR) and inserted into pMD18T simple vector, respectively. After being verified by sequencing, the hIL1Ra and HSV1tk were cloned into pMDIRES to construct pMDHSV1tkIREShIL1Ra. The HSV1tkIREShIL1Ra fragment was then cloned into pSNAV2.0. The recombinant plasmid pSNAV2.0HSV1tkIREShIL1Ra was constructed and identified by restriction enzyme. The recombinant plasmid was transfected into synovial cells with the Lipofectamine 2000 method. The mRNA level change of HSV1tkIREShIL1Ra was identified by reverse transcription polymerase chain reaction(RTPCR). ResultsThe recombinant plasmid was confirmed by restriction endonucleases digestion. The RTPCR results indicated that HSV1tk and hIL1Ra were effectually expressed in the transfected synovial cells. ConclusionThe recombinant plasmid pSNAV2.0HSV1tkIREShIL1Ra was successfully constructed and effectually expressed in the transfected synovial cells, which provided a sound base for gene therapy of osteoarthritis.

Key words: Thymidine kinase; Receptors, interleukin1; Synovial m embrane; Osteoarthritis