Construction and identification of recombinant vectors carrying herpes simplex virus thymidine kinase and cytokine genes expressed in gastric carcinoma cell line SGC7901

Abstract

Aim: To construct and identify the recombinant vectors carrying herpes simplex virus thymidine kinase (HSV-TK) and tumor necrosis factor alpha (TNF-alpha) or interleukin-2 (IL-2) genes expressed in gastric carcinoma cell line SGC7901.

Methods: The fragments of HSV-TK, internal ribosome entry sites (IRES) and TNF-alpha or IL-2 genes were inserted in a TK-IRES-TNF-alpha or TK-IRES-IL-2 order into pEGFP-N(3) and pLXSN to generate the therapeutic vectors pEGFP-TT, pEGFP-TI, pL(TT)SN and pL(TI)SN respectively, which were structurally confirmed by the digestion analysis of restriction endonuclease. The former two plasmids were used for the transient expression of recombinant proteins in the target cells while pL(TT)SN and pL(TI)SN were transfected into SGC7901 cells by lipofectamine for the stable expression of objective genes through G418 selection. The protein products expressed transiently and stably in SGC7901 cells by the constructed vectors were confirmed by fluorescent microscopy and Western blot respectively.

Results: The inserted fragments in all constructed plasmids were structurally confirmed to be consistent with that of the published data. In the transient expression, both pEGFP-TT and pEGFP-TI were shown expressed in nearly 50% of the transfected SGC7901 cells. Similarly, the G418 selected vectors PL(TT)SN and PL(TI)SN were confirmed to be successful in the stable expression of the objective proteins in the target cells.

Conclusion: The constructed recombinant vectors in the present study that can express the suicide gene TK in combination with cytokines genes may serve as the potential tools to perform more effective investigations in future for the gene therapy of gastric carcinoma.

Figure 1

Diagram of the construction of the expression vector PL(TT)SN.

Figure 2

Segment analyses of pEGFP-TI and pEGFP-TT by restriction endonuclease digestion. Lane 1: DNA marker (from top to bottom: 1 543, 994, 697, 515 and 337 bp); Lane 2: pEGFP-TI/EcoR I and Hpa I (TK gene 1 128 bp); Lane 3: pEGFP-TI/ EcoRI and XhoI (TK + IRES 1.71kb); Lane 4: pEGFP-TI/XhoI and BamH I (IL-2 gene 456 bp); Lane 5: pEGFP-TT/EcoRI and HpaI (TK gene 1 128 bp); Lane 6: pEGFP-TT/EcoRI and XhoI (TK + IRES 1.71 kb); Lane 7: pEGFP-TT/XhoI and BamHI (TNF-α gene 474 bp).

Figure 3

Transient expression of the recombinant genes in SGC7901 cells. A: Transient expression of pEGFP-TT in SGC7901 cells × 100. B: Transient expression of pEGFP-TI in SGC7901 cells × 100.

Figure 4

Stably expressed recombinant protein in transduced SGC7901 cells confirmed by Western blot. Lane 1: IL-2 (15ku) expressed in SGC/TI cells; Lane 2: TNF-α (17ku) expressed in SGC/TT cells.