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The Investigation on the Expression and Purification of Prokaryotic Protein of Human sTrail Gene Activited by uPA and the Activity of This Prokaryotic Protein Against NSCLC

Author: LiangYuJia
Tutor: XiongZuo
School: Third Military Medical University
Course: Internal Medicine
Keywords: Human tumor necrosis factor soluble -related apoptosis- inducing ligand gene the uPA cleavage point clone Antigenic Fusion protein Target protein Kill tumor activity of sTrail and
CLC: R734.2
Type: Master's thesis
Year: 2011
Downloads: 14
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Abstract

Background and Purpose: lung cancer, especially in non-small cell lung cancer, early diagnosis is difficult, about 80% is found for late surgery difficult undercutting treatment often postoperative chemotherapy. But traditional chemotherapy, the prognosis is poor, to a plateau, and has become the treatment of the \Standard first-line treatment, the effective rate of only 15% to 36% survival period of 8-10 months, 1-year survival rate of 30% to 40%. To break through this bottleneck to be the transformation of the mode of treatment. Personalized targeted therapy for this \In recent years, the rapid development of modern molecular biology techniques has brought new hope for the treatment of malignant tumors. Molecular targeted therapy intervention, individualized tumor treatment step onto a new level. A large number of clinical studies have fully confirmed the the tumor molecular targeted therapy theory correct and feasible. Targeted therapies ranging from first-line treatment, maintenance therapy and second-line treatment. But the attendant problems is how to overcome the resistance of the secondary beneficiary population how appropriate screening more people pay attention to, in particular, how to find new therapeutic targets. Tumor necrosis factor (tumour necrosis factor, TNF)-related apoptosis-inducing ligand (TNF-related apoptosis inducing of ligand Trail) genes present in the placenta, lung, kidney, spleen and peripheral lymphoid and other tissue cells, important induced tumor the role of apoptosis. Trail extracellular region (114 to 281 amino acid residues) can form of soluble the Trail molecules (sTrail), it widely caused by a variety of tumor cell apoptosis in vitro, but non-toxic side effects on normal cells, and induced apoptosis of tumor cells does not depend on whether the p53 molecule mutation. In, sTrail in cancer therapy with a broad application prospects. Urokinase-type plasminogen activator (urokinase plasminogen activator, uPA) is a glycoprotein containing 411 amino acids, the molecular mass of 50 to 60 kD. Normal tissue cells, the expression of uPA and its uPAR is very low, but almost all tumors, both significantly higher expression. uPA and tumor growth, invasion and metastasis and so closely related, therefore, be regarded as an important tumor marker and tumor attractive therapeutic target. Therefore, this study aims to explore containing the uPA cleavage site of human TNF soluble-related apoptosis-inducing ligand (TNF-sTRAIL) in vitro against non-small cell lung cancer activity of recombinant target protein for further study in vivo of lung cancer targeted treatment lay the foundation. Methods: In this study, using genetic engineering techniques to sequence the tandem His peptide sequence with uPA cleavage site (SGRSA) to introduce people sTrail C-terminal to protein molecular conformation change, so that by His peptide with uPA cleavage sites closed No activity (or lower activity), the fusion molecule, and its specific composition order, from the N-end to C-terminal order: the sequence of the cleavage site of uPA in the mature sTRAIL → → multi series His peptide. Cloned into the sTrail-uPA-his fusion gene and subcloned into the prokaryotic expression vector pET-32a induced expression vector tag (Trx) fusion protein, EK digestion to its label proteins was the target protein sTrail-uPA. Purification of the target protein underwent immunogenicity identification. Vitro experiments confirmed the uPA enzyme cleavable sTrail-uPA release with kill tumor activity sTrail, as follows. One containing the uPA cleavage site the people sTrail gene original nucleoprotein expression and purification of 1. The people sTrail gene cloning and its original nuclear the plasmid pMDl8-T/sTrail the design to amplification sTrail gene primers, human placental tissue total RNA as a template length cDNA sequence, RT-PCR was amplified sTrail gene construct prokaryotic plasmid pMDl8-T/sTrail the sequencing confirmed whether cloned into the full-length coding sequence of sTrail gene. 2. Of with uPA cleavage point sTrail gene of the original construction of nuclear expression plasmid pET-32a/sTrail-uPA-his of the design PCR method to build sTrail-uPA-his fusion gene primers, to pMD18-T/sTrail template, primer extension law, and further build the prokaryotic expression plasmid containing the reorganization of of uPA cleavage site (SGRSA), sTrail gene. The sequencing confirmed whether successfully constructed prokaryotic expression vector carrying sTrail gene pET-32a/sTrail-uPA-his reorganization. The 3. Induced expression and purification of sequencing recombinant fusion protein sTrail-uPA-his the plasmid pET-32a/sTrail-uPA-his transformed expression strain BL21 (DE3). Pick-positive clone culture, 28 ℃, IPTG overnight induction. SDS-PAGE analysis is correct, whether the expression product to detect the expression level. Charge to induce bacterial, resuspended bacteria, sonicated, effect of microscopy broken bacteria. Specimens sonicated supernatant and the precipitate filtered supernatant. The Ni2 column in the metal chelating aggregate into the filler. Supernatant sample, to collect penetrating fluid, complex balancing, respectively, with a 5%, 10% and 100% Elution Buffer eluting, 100% component dialysis in Tris buffer. Purified by SDS-PAGE electrophoresis detects whether to the fusion protein. Fusion protein cleavage 4.sTrail-uPA-his purity detection and immunological identification of the fusion protein solution EK enzyme overnight digested specimens uncut sample as a control, the SDS-PAGE electrophoresis whether to cut the target protein tagged protein. The digested target protein by high performance liquid chromatography (high performance liquid chromatogram, HPLC) analysis, the normalization method knowing purity. The target protein after electrophoresis transferred to nitrocellulose membranes soaked in containing human Trail sheep anti-blocking solution, overnight incubation, plus horseradish peroxidase-labeled rabbit anti-sheep secondary antibody, incubated with the chemiluminescent substrate, camera The coexist Figure. Whether this identification of the target protein with Trail antigenicity. Containing uPA cleavage site the people sTrail gene original nucleoprotein activity against non-small cell lung cancer 1. STrail-uPA recombinant protein in cleaved by uPA vitro of tumoricidal IC50 calculation NCI-H460 human non-small cell lung cancer cells were seeded at 96 well plates (3000 cells / 150μl / holes), 37 ° C, 5% CO ₂ sTrail-uPA environment for 24 h, different concentrations of purified recombinant protein and uPA enzyme were only plus sTrail recombinant protein-uPA treated cells of uPA enzymes as control for 24 h, and joined CCK-8 (cell counting kit-8) were cultured for 4 h reagents under the same conditions, efficient ultraviolet analyzer optical density at the wavelength of 450 nm value, calculated knowing IC ₅₀. 2. STrail-uPA recombinant protein non-small cell lung cancer cell apoptosis in vitro inoculation of 1 × 106/ml logarithmic phase of NCI-H460 cells, cultured for 24 h, were added to different final concentrations of recombinant protein sTrail-uPA the uPA enzyme, under the same conditions were cultured for 24 h and 48 h, NCI-H460 cells alone as a control. The cells were collected, treated with Annexin V-PI double staining, tumor cell apoptosis was detected by flow cytometry. In order to detect the target protein sTrail-uPA after the uPA enzyme vitro lysis ability to release with the killing activity sTrail induced apoptosis of NCI-H460 non-small cell lung cancer cells, and whether the apoptosis-inducing with dose - dependent \dependent characteristics. 3 sTrail-uPA recombinant proteins for non-small cell lung cancer cell growth - growth curves drawn in H460 cells inoculated into four 96-well plates (3000 cells / 150μl / holes), cultured 24 h after sTrail-uPA protein the uPA enzyme, respectively, only sTrail-uPA protein, pure uPA enzyme in H460 cells and H460 cells alone as control simple culture medium for the control, respectively, for 24 h, 48 h, 72 h, 96 h Remove a 96-well plate added to each well get a new medium with CCK-8, 4 h, under the same conditions to continue to foster and shaken for 30 seconds and mix efficient ultraviolet analyzer optical density at a wavelength of 450 nm, the optical density value of the Y-axis, time The X axis plots Enhanced curve. This is detected by the the uPA enzyme in vitro cleavage sTrail-uPA target protein to inhibit the activity of tumor cell growth. Results: First, people sTrail gene containing the uPA cleavage site of the original nuclear protein expression and purification of sequencing confirmed, the clone to sTrail gene is encoded sequence, and successfully constructed prokaryotic plasmid pMDl8-T/sTrail. 2. Sequencing confirmed the the carry-containing uPA cleavage site (SGRSA), sTrail gene pET-32a/sTrail-uPA-his reorganization of prokaryotic expression vector can be successfully constructed. 3. The recombinant plasmid pET-32a/sTrail-uPA-his by induced expression of a fusion protein expression levels accounted for 55% of the total bacterial proteins. The fusion protein by the metal chelating Ni ^{2 column purification, SDS-PAGE electrophoresis molecular weight of approximately 38 kD. Purified fusion protein digested by EK, respectively cut approximately 19.5kD target protein sTrail-uPA and 18.5kD Trx-His-tagged proteins. Digested target protein line HPLC analysis, normalization method knowing purity of 98.6%. 6 Western blot of the Objective protein line the immunological identification results in about 19.5kD stained bands, visible target protein with Trail antigenic. UPA cleavage site containing human sTrail gene original nucleoprotein against non-small cell lung cancer activity of sTrail-uPA recombinant protein by the the uPA enzyme in vitro lysis can be released with killing activity of sTrail and NCI-H460 human lung cancer cells, the anti- activity in a dose - response relationship, with an IC50 of 20 ng / ml. Flow cytometry, 25 ng / ml of recombinant protein sTrail-uPA and uPA enzyme NCI-H460 cells 24 h and 48 h, apoptosis rates were (8.85 ± 1.36)% and (22.74 ± 3.18) %, both compared to the difference statistically significant (P lt; 0.05); 100 ng / ml recombinant protein sTrail-uPA and uPA enzyme NCI-H460 cells apoptosis rate of 48 h (34.95 ± 3.09)% with the same duration of action of 25 ng / ml of recombinant protein sTrail-uPA compared with the uPA enzyme action NCI-H460 cells, the difference was statistically significant (P lt; 0.01). The prompt restructuring the purpose of protein sTrail-uPA by the the uPA enzyme in vitro lysis release with killing activity sTrail induced apoptosis of NCI-H460 non-small cell lung cancer cells, and the apoptosis induction with time - dependent and dose - dependent \. STrail-uPA reorganization purpose protein of non-small cell lung cancer cell growth curve known sTrail-of uPA recombinant protein by uPA enzyme in vitro lysis release with cytotoxic activity of sTrail human lung cancer NCI-H460 cell growth with significant with killing effect ( Compared with the control cells and the cells of the uPA enzyme treatment, P LT; 0.01; cultured for 2-4 days, after the treatment sTrail-uPA protein alone treated cells comparison, P LT; 0.05). Conclusion: 1. Cloned into the fusion gene containing uPA cleavage site sTrail-uPA, and successfully constructed prokaryotic expression vector pET-32a/sTrail-uPA-his.2. Digested, purified carrier pET-32a/sTrail-uPA- his by the induction of the expression of the fusion protein sTrail-uPA-his, the purity of 98.6% and a molecular weight of 19.5 kD the target protein sTrail-uPA. 3. Target protein sTrail-uPA the uPA enzyme vitro cleavage releases with killing activity of sTrail and tumor cells with an IC50 of 20 ng / ml. The cytotoxic activity of sTrail could significantly inhibit the growth of human lung cancer NCI-H460 cells, significant induction of apoptosis in the tumor cells.}

The Investigation on the Expression and Purification of Prokaryotic Protein of Human sTrail Gene Activited by uPA and the Activity of This Prokaryotic Protein Against NSCLC

Abstract

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