ABSTRACT
Objective: To investigate the inhibitory effect of eukaryotic expression vector (attenuated salmonella typhimurium) carrying tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and Chicken anemia virus VP3 gene on gastric cancer cells in vitro and in vivo. Methods: The cloning vectors pBud-TRAIL, pBud-VP3, and pBud-TRAIL-VP3 were transformed into attenuated Salmonella typhimurium by electric transformation technique. The S. typhimurium-based carriers were then transfected into gastric cancer cells, line SGC-7901 after stability assay. The expression of fusion green fluorescent protein was examined using fluorescent microscopy after 24 h. MTT assay was used to examine the inhibition of cell growth. Flow cytometry was used to detect cycle distribution and apoptosis rates of cells. The expression of caspase-3 and caspase-9 was assayed by immunohistochemistry method. Salmonella typhimurium carrying recombinant plasmid was administrated orally in sarcoma-bearing mice; 8 weeks later RT-PCR was used to detect the expression of cloning vectors in tumor tissue. Meanwhile, the sizes of tumors were also determined. Results: The recombinant plasmids were stably transformed into attenuated Salmonella typhimurium, and the plasmids was satisfactorily expressed in gastric cancer cells via attenuated Salmonella typhimurium. TRAIL and VP3 inhibited the proliferation of gastric cancer cells after 48 h. Flow cytometry analysis showed that the pBud-TRAIL-VP3 obviously enhanced apoptosis rates of gastric cancer cells. TRAIL and VP3 jointly increased the expression of caspase-3 and caspase-9. In vivo study showed that TRAIL and VP3 genes were expressed in tumor tissue and could inhibit the tumor growth(P<0.05). Conclusion: Attenuated Salmonella typhimurium-mediated TRAIL and VP3 transfection of gastric cancer cells can inhibit cell growth in vitro and in vivo. The joint effect of TRAIL and VP3 is correlated with the increase of caspase-3 and caspase-9 expression.
ABSTRACT
Objective:To construct the extracellular region of the human TRAIL cDNA expression vector and express and purify the extracellular region of the TRAIL protein. Methods: The mRNA of TRAIL was extracted from CD3 activated normal human PBMC and used as a template for reverse transcription. After PCR amplification, a 730 bp fragment including extracellular region was obtained and cloned into pGEX-2T.The recombinant vector was named pGEX/TRAILex. The pGEX/TRAILex vector was transformed into E.coli DH5a. After IPTIG induced at lower temperature, the collection of the sonicated extract was purified by using the GST agarose 4B. The purified fusion protein was identified by Western blotting with anti-TRAIL McAb.Results:The pGEX/TRAILex was constructed. After IPTG induced,a high level expression of the extracellular region of the TRAIL protein was obtained, SDS-PAGE analysis showed that the recombinant E. coli could express a 54 kD GST fusion protein which accounted for about 28% of the total cellular protein. The study of solubility of expression protein indicated that GST-Tex was expressed predominantly in the soluble form.The purified production was obtained 2.2 mg/L of culture media and the purity of the GST-Tex was more than 95%. GST/TRATLex protein could be recognized by anti-TRAL McAb in Western blot. Conclusion:The expression of recombinant extracellular domain of the human TRAIL protein may be useful for the study of biological functions of TRAIL and it's biotheraphy in tumor.
ABSTRACT
Objective To evaluate the expression and activity of GFP/TRAIL gene activated by the hTERT promoter on colon cancer cell line DLD-1. MethodsGFP/TRAIL gene activated by the hTERT promoter was transfected into DLD-1 with adenoviral vector,expression and apoptosis inducing ability of GFP/TRAIL protein was determined by fluorescence-activated cell sorting (FACS). [WT5”HZ]ResultsThe expression of GFP gene is 41.63% and 54.07% with either hTERT promoter or CMV promoter in DLD1 cells;GFP/TRAIL gene was able to inhibit cell growth(93.50%) and induce apoptosis(56.97%) of DLD-1 ,there was significant difference between Ad/hTERT-gTRAIL and the other two control groups (PBS and Ad/hTERT-LacZ)( P