A Latent Soluble TNF Receptor Type I (sTNFRI) Fusion Protein In Targeting Treatment Of Endometriosis

ObjectiveTo engineer a latent sTNFRⅠusing Latency Associated Protein (LAP) of Transforming Growth Factor-β1 (TGF-β1) fused via a Matrix Metalloproteinase (MMP) cleavage site to soluble Tumor Necrosis Factor ReceptorⅠ( sTNFRⅠ). Then the latent biological activity and activation specificity of the fusion protein were evaluated, which would open up a new idea on targeting treatment of endometriois.Methods1. A double-stranded deoxyoligonucleotide coding for MMP cleavage site was cloned into plasmid pcDNA3.1(+). Then to construct the recombinant vector pcDNA3.1/LAP- MMP-sTNFRⅠ, the TGF-β1-LAP and sTNFRⅠcDNA were inserted into the two sides of MMP cleavage site respectively.2. After nucleotide sequencing, the recombinant vector pcDNA3.1/LAP-MMP-sTNFRⅠwas transfected into COS-7 cells by Lipofectamine 2000TM,and the expression of fusion gene in COS-7 cells was detected by RT-PCR and Western Blotting.3. The sTNFRⅠbiology, which was assessed on mouse L929 cells by inhibition of cytopathic effect of TNF-α, was performed to evaluate the latent biology activity of the fusion protein. Supernatants of transfected cells were collected for sTNFRⅠbiology assay with or without incubation of MMP or peritoneal fluid from EMs patients.Results1. The results of restriction endonuclease digestion and sequencing demonstrated that the recombinant plasmid was constructed successfully. Transient expression of fusion gene LAP-MMP-sTNFRⅠwas confirmed by RT-PCR and Western Blotting.2. sTNFRⅠbiology assays showed no significant change between death rate of L929 cells of pcDNA3.1/LAP-MMP-sTNFRⅠ-transfected supernatants and pcDNA3.1- transfected supernatants (P>0.05). But incubation of either MMP-2 or peritoneal fluids from EMs patients to supernatants from pcDNA3.1/LAP-MMP-sTNFRⅠ-transfected COS-7 cells significantly decreased the death rate of L929 cells (P<0.05).ConclusionThe recombinant eukaryotic expression vector pcDNA3.1/LAP-MMP-sTNFRⅠhas been constructed and expressed successfully in COS-7 cells. The LAP-MMP-sTNFRⅠfusion protein lacks biological activity until incubated with MMP or peritoneal fluid from EMs patients, contributing to further studies on targeting treatment of endometriosis.