Cloning,Expression,Purification Of RGD-sTRAIL And Its Anti-tumor Activity In Vitro And Targeting To Tumor In Vivo

Objective To construct the recombinant vector which can express soluble human TNF-related apoptosis-inducing Hgand(sTRAIL, 114-281 amino acid) fused with RGD motif (Arg-Gly-Asp) in E.coli,express and purify RGD-sTRAIL.We investigated the antitumor activity of RGD-sTRAIL in vitro and the distribution of RGD-sTRAIL in tumor in vivo. Methods PCR was used to amplify soluble hTRAIL cDNA fused with RGD motif,and RGD-sTRAIL cDNA was ligated into the pGEM3Zf(-) vector.The recombinant plasmid was transformed into DH5a.Positive clones were screened by blue/write clonesAfter identified by restrictive endonuclease mapping and DNA sequencing,the recombinant plasmid was amplified in the host cells.The recombinant plasmid was digested by the restriction endonuclease(NdeI and SalI).The target gene,RGD-sTRAIL,was inserted into pET11a to construct the expression vector.The recombinant plasmid was transformed into BL21.The pET11a-RGD-sTRAIL/BL21 was induced by IPTG and RGD-sTRAIL protein was analysed by SDS-PAGE and identified by Western blotting. In order to obtain active RGD-sTRAIL protein,inclusion bodies should be denatured and then renatured.After refolding,RGD-sTRAIL was purified by CM-Sepharose FF and Sephcryl S-100After treating the human lung carcinoma cell line A549 with different concentrations of RGD-sTRAIL,we observed the shrinking of the cells.The antitumor activity was proved by crystal violet staining method.The purified RGD-sTRAIL protein was injected intraveneously into tumor-bearing mice.The tumor tissues were cut at different time after injection and we can observe the distribution of the proteins by immunohistochemiostry. Results The resultsdemonstrated that the sequence of RGD-sTRAIL gene was correct and the target gene was correctly inserted into pET 11 a. Results showed that RGD-sTRAIL was expressed in 20kD in BL21 and the expression level accounted by 20% of the whole protein of the bacteria.Most of the target proteins were expressed as inclusion bodies.The recombinant protein could specifically react with a rabbit monoclonal IgG antibody against human TRAIL by Western blotting.The purity of the RGD-sTRAIL protein was about 95% after purification.The A549 was treated by RGD-sTRAIL and morphological shrinking was observed under optical microscope,IC50 is about 1.48 ng/ml.The distribution of RGD-sTRAIL in endothelial cells of tumor vasculature was higher than that of TRAIL. Conclusions The expressed plasmid was correctly constructed,RGD-sTRAIL was expressed in E.coli,and the RGD-sTRAIL was refolded and purified successfully.The refolded and purified RGD-sTRAIL can apparently inhibited the growth of the cells of A549 in dosage-depent relationships in vitro.In vivo,RGD-sTRAIL can target to vasculature in tumor tissues.