Aim: To develop a gene therapy vector of interleukin-1 receptor II(IL-1RII) with recombinant adenovirus and express IL-1RII in the eutopic stromal cells of endometriosis(EM).
Methods: Get full length of cDNA with IL-1RII gene was obtained by PCR. The gene was then subcloned into the pShuttle-CMV shuttle vector. The resultant plasmid (pShuttle-CMV-RII) was cotransduced into E.coli BJ5183 cells with pAdEasy-1 plasmid to undergo homologous recombination by electroporation. The linearized recombinant plasmid (pAd-RII) was transfected into 293 cells. The recombinant adenovirus was detected by examining the expression of IL-1RII while the recombinant adenovirus of LacZ gene was constructed as control. The stromal cells of EM were infected by the recombinant adenovirus and the expression of IL-1RII was detected by immunohistochemistry(IHC).
Results: It was confirmed by sequencing that the two ligand products had no mutation of IL-1RII. Restriction endonuclease analysis confirmed the successful cloning of the gene into the pShuttle-CMV and the recombinants(pAd-RII) were selected for kanamycin resistance. Presence of the recombinant adenovirus was confirmed by the X-gal stain of LacZ and the expression of soluble IL-1RII was detected by ELISA. IL-1RII was expressed in the stromal cells of EM by IHC.
Conclusion: The recombinant adenovirus of IL-1RII has been successfully constructed and expressed in the stromal cells of EM, which provides a basis for the research into the role of IL-1RII and the effect of biological treatment in EM.