Construction And Expression Of Stx1A-LHRH Fusion Toxin

Recombinant toxin is a kind of biological missiles, which can target tumor cells and kill them, and it will have a good using prospect in tumor therapy. So recently it becomes one of the hotspots of tumor target therapy. The recombinant toxins consist of recognition system and kill system. Recognition system has strong target function at tumor cells, and it can specific recognize tumor cells. This kind of oriented parts usually are monoclonal antibodies,gene engineering antibodies or cytokine。Kill system have the function of kill and wound cells, usually using bacterial toxin or plant toxin to carry out ,for example diphtheria toxin,castor toxin . Through the cooperation of recognition system and kill system we can specific kill and wound target cells. Shiga toxin is a protein toxin produced by Enterohemorrhagic E.coli O157:H7. It includes two subtypes: Stx1 and Stx2. Stxs consist of an A-chain in noncovalent association with a pentamer of B-chains, and it belongs to the AB family of protein toxins. The A subunit can be cleaved by trypsin, separating it into the A1 fragment and the A2 fragment. The A1 subunit has N-glycosidase activity, as well as the A2 subunit is a bridge between the A1 and B subunits. The pentamer of B subunits binds to receptors on cells'surface, and mediates the entry of the A subunit into cells and producing biological activity. And the receptor of shiga toxin is globotriaosylceramide (Gb3). After binding with these receptors, Stx is internalized by receptor-mediated endocytosis, delivered to the endoplasmic reticulum, then transported to the cytoplasm where A and B subunits are separated. A subunit is separated into the enzymatically active A1 fragment and the A2 fragment by trypsin. The A1 fragment inhibits protein synthesis and produces cell apoptosis. This function can result in the death of Vero cell,Hela cell or any other cells with Gb3 receptors. Recent study demonstrates that the immunotoxin designed with intact shiga toxin has some nonspecificity, because the B subunits can bind with Gb3 receptors on the surface of normal cells and induce the cells death. Study suggests that the intact A subunit hasn't the largest enzymatic activity because the presence of the A2 fragment has an inhibitory effect on the enzymatic activity of Shiga toxin .So we only amplify some amino acids residues sequence of the A subunit without signal peptide. Hypothalamic Gonadotropin-Releasing Hormone (GnRH) can stimulate the pituitary synthesis and secret FSH and LH, which can specific bind with GnRH receptors. Study indicates LHRH (luteinizing hormone-Releasing Hormone) can control both FSH and LH, so LHRH and GnRH are the same substance. GnRH receptors primarily exist in gonadotroph of hypothalamo-pituitary axis, and they also express in gonadal tissues. Study indicates that there are GnRH specific binding sites on the surface of cancer cells, especially hormone-dependent cancer cells, for example carcinoma of endometrium ,epithelial ovarian tumor,breast cancer and so on.Recently data shows some non-hormone dependent tumor, such as pancreas,kidney cancer and hepatocellular carcinoma also react to the GnRH analogues. So we consider it is a wide existed phenomenon to the majority of adenocarcinoma cells, including lung,breast,ovary,kidney,liver,endometrium,cervical adenocarcinoma. So we construct the recombinant shiga toxin. We designed two primers to amplify the fragment of Stx1A from EHEC O157:H7 by PCR, and fused to structure sequence of LHRH. And thus Stx1A-LHRH fusion gene was constructed. After digested with restriction endonuclease NcoI and EcoRI, the fusion gene was orientally inserted into expression vector pET28a. The resultant recombinant plasmid, terming pET28a:: Stx1A-LHRH, was transformed into E. coli BL21(DE3). IPTG was used to induce the expression of the target protein. After the harvested bacteria were dispersed with sonication, the inclusion bodies were extracted with centrifugation. SDS-PAGE was used to analyze the expression of fusion protein. The recombinant Stx1A-LHRH fusion protein was high level expressed in inclusion bodies of E. coli and covered above 37.6% of total bacterial proteins. Induce time ranging from three hours to seven hours, the expressing of fusion protein hasn't obvious changed. Because renaturation is a very complex course, which is determined by many factors, and the renaturation conditions of different proteins are different, we need many experiment to find the suitable method. So we refold the inclusion bodies and try to make the fusion protein soluble express at the same time. We designed...