Construction of AdCTLA4Ig-IRES-OX40Ig and Inhibition Activity of Its Expression Products on MLR in vitro

WANG Guang-Ming, YANG Yang, LI Ai-Ling, HAO Jie, GAO Xiang, XIE Shu-Sheng *

Chinese Journal of Biochemistry and Molecular Biology ›› 2005, Vol. 21 ›› Issue (05) : 597-602.

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Chinese Journal of Biochemistry and Molecular Biology ›› 2005, Vol. 21 ›› Issue (05) : 597-602.
Article

Construction of AdCTLA4Ig-IRES-OX40Ig and Inhibition Activity of Its Expression Products on MLR in vitro

  • WANG Guang-Ming, YANG Yang, LI Ai-Ling, HAO Jie, GAO Xiang, XIE Shu-Sheng *
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Abstract

The vector pT/CTLA4Ig-IRES-OX40Ig was constructed. The vector pT/CTLA4Ig-IRES-OX40Ig and pAdeasy-1 were cotransfected into competent BJ5183 E.coli by electroporation. pAdCTLA4Ig-IRES-OX40Ig were subsequently transfected into 293A cells with LipofectinAMINE2000 to generate AdCTLA4Ig-IRES-OX40Ig,which is able to simutaneously express both of the fusion proteins CTLA4Ig and OX40Ig, respectively. The adenovirus containing CTLA4Ig and OX40Ig genes was propagated and purified. The titer of the stock virus was assessed using 293A cells and the expression of CTLA4Ig and OX40Ig was confirmed in culture supernatant of 293A cells using Western blot. Its inhibition activity was evaluated through mixed lymphocyte reaction (MLR) using the isolated Lewis spleen cells infected with AdCTLA4Ig-IRES-OX40Ig as stimulator cells and the BalB/c spleen cells as responder cells in vitro. The restriction map confirmed that the vector pT/CTLA4Ig-IRES-OX40Ig was correctly constructed and obtained the recombinant AdCTLA4Ig-IRES-OX40Ig. The results indicated that the conditioned supernatant of 293A cells transfected with the AdCTLA4Ig-IRES-OX40Ig contained both of the fusion protein CTLA4Ig and OX40Ig.The results of MLR demonstrated that transfection of AdCTLA4Ig-IRES-OX40Ig was capable of inhibiting xenogeneic MLR by a way that much stronger than that of AdCTLA4Ig and AdOX40Ig alone. A greater in vitro MLR inhibition effect of AdCTLA4Ig-IRES-OX40Ig transfected rat spleen cells was also demonstrated.

Key words

CTLA4Ig / OX40Ig / internal ribosome entry site

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WANG Guang-Ming, YANG Yang, LI Ai-Ling, HAO Jie, GAO Xiang, XIE Shu-Sheng *. Construction of AdCTLA4Ig-IRES-OX40Ig and Inhibition Activity of Its Expression Products on MLR in vitro[J]. Chinese Journal of Biochemistry and Molecular Biology, 2005, 21(05): 597-602

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