The Prokaryotic Expressing And Purification Of Recombinant Protein TriTH/Cripto-1_29-164/TSP50_114-349

As the major killer of the people of the world,malignant tumor has been a serious threat to people's life and health.The traditional treatments of malignant tumor,including chemotherapy,surgery,radiotherapy,have several disadvantages during the clinical treatment,like serious side effects,recurrence and failed in against tumor immune tolerance.Immunotherapy is a new approach for malignant tumor treatment with good application prospect.And the tumor fusion protein vaccines,which have low toxicity and can break immune tolerance,also get wide spread attention in medical community.The key to developing tumor vaccine lies in the selection of tumor antigen target that have good immunogenicity and can specifically targeting tumor cells.Carcinoembryonic antigen?CEA?is a tumor-associated antigen that widely expressed in many malignant tumors.The safety of the CEA protein vaccine has been confirmed.Due to the weak immunogenicity of full length CEA protein,it is supposed to connect the triple repeated Th epitope peptide of CEA?CEA625–677,triTH?with other tumor antigens.As a tumor-associated antigen,Cripto-1 expressed ubiquitously in more than 10 different types of cancers,but not or faintly expressed in normal adult body tissue.Recent studies found that Cripto-1 can promote tumor proliferation,infiltration,and metastasis as an oncogene.Cripto-1 includes two key functional domains,EGF-like domain?aa75-112?and CFC domain?aa114-150?.The last antigen is testes-specific protease 50?TSP50?,which is a cancer testis antigen that not expressed or low expressed in normal adult tissue,but highly expressed in human testes tissue and a wide variety of tumor tissue.Previous research has shown that as a proto-oncogene,TSP50 can promote tumor development and progression by activating NF-kB signaling pathway via its catalytic triad domain?aa114-349?.In order to prepare tumor protein vaccines with high specificity and strong immunogenicity,we constructed triTH-TSP50_114-349 and triTH-Cripto-1_29-168 fusion protein expression vectors and expressed them in E.coli.In this study,we constructed the pET28a-Cripto-1_29-168 and pET28a-triTH-Cripto-1_29-168prokaryotic expression plasmids,and the pET28a-triTH,pET28a-TSP50_114-34914-349 and pET28a-triTH-TSP50_114-34914-349 plasmids were provides by our laboratory.These five plasmids were transformed into BL21 competent cells and induced expression by IPTG.SDS-PAGE and Western blot analysis were conducted to analyze the expression of these fusion proteins.The results showed that these recombinant proteins can successfully and correctly expressed in BL21 bacteria except triTH-TSP50_114-349.Our study implied that the pET28a-triTH-TSP50_114-34914-349 plasmid has losing its His-tag,so in the follow-up experiment,we reconstructed the pET28a-triTH-TSP50_114-349 plasmid and expressed the recombinant protein successfully.In order to get enough protein,we optimized the expression conditions of triTH-TSP50_114-349andtriTH-Cripto-1_29-168.Theformerrecombined protein expression peaked in induction condition of 0.05mM/L IPTG inducing 5h while the latter fusion protein is 1mM/L IPTG inducing 1h.Then we expressed these five fusion proteins abundantly.After washed and solubilized by denaturation buffer,we pured these inclusion bodies by HisTrap FF crude.At last we refolded these denatured protein by dialysis or dilution.In the follow-up experiment,we detected the effect of 5 fusion proteins on T lymphocyte transformation,the results showed that all of these fusion proteins especially triTH-TSP50_114-349 and triTH-Cripto-1_29-168 can accelerate the T lymphocyte transformation.These results suggest that the fusion proteins of triTH-Cripto-1_29-168 and triTH-TSP50_114-349have immunogenicity in vitro.This study laid a important material base for development of tumor vaccine.