Cloning, Expression, And Characterization Of Human Tumor Necrosis Factor-like Weak Inducer Of Apoptosis

[Background]Malignant tumors are common diseases which severely harm people's health. The discovery and anti-tumor effect of tumor necrosis factor (TNF) family provide effective ways for prevention and treatment of malignant tumors. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) was first cloned by Chicheportiche, et al. in 1997. It was named as TNF ligand superfamily member 12(TNFSF12) . It is a type II membrane protein and is expressed in a wide-range of tissues and cells. It can mediate various biological effects including apotosis, proinflammation, regulation of immunity and angiogenesis. The recombinant soluble TWEAK expressed in insects can induce apoptosis in human colon adenocarcinoma HT-29, human gastric adenocarcinomaKATO-III and human oral squamous cell carcinoma HSC3 cells in the presence of INF-y. The soluble TWEAK expressed in HEK293 cells can kill rhabdomyosarcoma Kym-1 cells. The signaling pathways of TWEAK-induced apoptosis are not very clear. Marsters, et al. found that TWEAK binds to the death domain-containing receptor 3 (DR3), induces apoptosis, activates NF-KB in human cell lines, and has overlapping signaling functions with TNF. Recently fibroblast growth factor-inducible 14(Fn14) has been identified to be a TWEAK receptor. The apoptotic cell death pathways include caspase-dependent apoptosis, cathepsin B-dependent necrosis, and endogenous TNF-α-mediated cell death. On the basis of studies on TNF-α, TRAIL, etc. in our research group, here we report firstly the cloning, expression, and biological activities of TWEAK in China.[Objective](1 )To clone the entire coding sequence and extracellular domain of TWEAK from human tissues.( 2 ) To express the extracellular domain in a fusion protein in E. coli and purify it.( 3) To examine the apoptotic death of transformed and tumor cells after treatment with recombinant sTWEAK and explore its possible mechanisms.(4) To construct recombinant adeno-X virus DNA-TWEAK vector and analyse its expression in HEK293 cells. [Methods](1) The entire coding and extracellular region genes of TWEAK were amplified by RT-PCR . The PCR products were examined by agarose gel electrophoresis. The target gene fragments were purified by gel extraction kit and ligated to cloning vector pMD18-T. The recombinant vectors were transformed into host strain E. coli K802 by lithium chloride method, screened and identified with PCR and restrictive enzymatic digestion. Their sequences were confirmed by DNA sequencing.(2) sTWEAKl gene was subcloned into expression vector pProEx HTb and transformed into E. coli BL21. sTWEAK2 gene was subcloned into expression vector pMAL-C2x and transformed into E. coli TB1. The recombinant vectors were screened and identified with PCR and restrictive enzymatic digestion. The recombinant fusion proteins were induced to express with IPTG, detected by coomassie brilliant blue-stained SDS-polyacrylamide gel electrophoresis(SDS-PAGE), and confirmed by Western blot analysis.(3) The sTWEAKl fusion protein was purified with Ni-NTA Spin Kit.(4) The biological activity was assayed on transformed and tumor cells by microplate photometer after crystal violet or sulfur rodamine B staining.( 5 ) The contents of IL-8 in the supernatant of 1990 cell cultures were determined by ELISA.(6) The morphological changes of the sensitive cells were observed by light and transmission electron microscopies.(7) The cell cycle and apoptotic rate were assayed by flow cytometry in 1990 and M85 cells.(8) The effect effusion proteins on induction of NF-kB in 1990 and LOVO cells was detected with Dual-Luciferase Reporter Assay system.(9) The TWEAK gene was subcloned into Adeno-X Viral DNA with pShuttle vector and transfected into HEK293 cells by lipofectamine method. The recombinant vector was identified by PCR.[Results](1) The entire coding and extracellular region genes of human TWEAK were successfull...