The Effect Of Diphtheria Toxin/Human B-cell Activating Factor Fusion Protein On Human Acute Lymphoblastic Leukemia BALL-1Cells:an Experimental Study

PART1Expression, identification and purification of Diphtheria toxin/human B-cell activating factor fusion proteinBackground:The concept of targeted therapy was presented as early as in1956by the German chemist Ehrlich. Among the current anticancer drugs, targeted cancer therapy because of its specificity and fewer side effects and other advantages have become one of the important research areas, its main mechanism:the specificity of the targeting vector and toxic substances through a certain method to connect targeting vector directed targeted therapy drug delivery to the tumor site, the toxic substances in killing tumor cells. Toxic substances, also known as "warheads, include toxins, radioisotopes and chemical drugs. Tumor targeting immunotoxin therapies targeting due to its high specificity and toxins destruction strong characteristics is the hot area of targeted cancer therapy research.In the1980s, researchers basing on the concept of targeted therapy had produced the first generation of immunotoxins, using the method of chemical coupling, the toxin protein by disulfide bonds or thioether bond being conjugated to a targeting vector.In animal tests it show low systemic side effects and targeted killing characteristics. Commonly, coupling agent includes: N-succinimidyl-3-[2-pyridyldithio]-propionate(SPDP),N-hydroxy-succinimidyl, S-acetyl-mercapto-succinic anhydride, and so on. With the development of molecular biology techniques, the second generation of immunotoxins came into being, that targeting vector with toxin fusion construct recombinant immunotoxins by recombinant technology. Researchers also by genetic recombination technology, are improving characteristics of the toxin, such as:removal of the cell binding domain in toxin structure to decrease its non-specific toxicity, and to improve its anti-tumor cells by a gene mutation. On the other hand, according to the diversity of the target antigen, the researchers are having used molecular biology techniques to build a diversity of carrier. To transform immunogenicity and toxicity are promoting the development of immunotoxins research.The types of toxic molecules to build immunotoxin are many, main categories: vegetable toxins, bacterial toxins, human derived protein toxins, fungal toxins. Diphtheria toxin (DT) are bacterial toxins, is common one of toxic molecules to construct the immunotoxin toxin molecules. DT is β phage gene encoding exotoxin after diphtheriae was lysogenized byβ phage, its molecules full-length being535aa and its molecular weight being58330D.DT was divided into3independent of each other domains:activity zone (C zone), the transmembrane transporter zone (T zone) and membrane receptor binding domain (R area), to facilitate the split operation, for designing the immunotoxin provide to present favorable conditions.Using molecular biology techniques we can produce truncated DT (such as DT388, DAB389, etc.) or the T zone to remove or point mutations, generating a series of DT derivatives or DT mutant (e.g. CRM9, CRM107).They are mostly retained C and T district will retain the activity and function of transmembrane transport. Through recombinant technology R mutants inactivated or deleted, using the structures, such as cytokines, monoclonal antibodies etc, which can specifically recognize the tumor cell surface molecules, replace R to connect the diphtheria toxin to construct immunotoxin, both targeted binding to tumor cells, but also the targeted killing of tumor cells, and low toxicity to normal cells. Based on the tumor cell surface expression of specific receptors to select carrier for the build immunotoxin, the option consist of binding antibodies, cytokines, hormones, etc. B lymphocyte activating factor (BAFF), being a kind of cytokine, can be combined with its expression of the receptor in the cell surface of B cell lines, therefore to be suitable as immunotoxins targeting vector.Based on this idea, this study is to utilize truncated DT388and B lymphocyte activating factor (BAFF) to construct recombinant immunotoxins DT388sBAFF through recombinant technology.Purpose:Constructing the immunotoxin diphtheria toxin/B cell activating factor fusion protein (DT388sBAFF) expression vector, inducing expression was identified and purified.Methods:1. Diphtheria toxin/human B cell-activating factor (DT388sBAFF) optimized synthetic gene primers were designed by PCR amplification DT388sBAFF inserted into the cloning vector pMD19-T after colony PCR, restriction enzyme digestion and DNA sequencing positive clones.2. After the target gene positive cloning vector pMD19-T-DT388sBAFF by double digestion (NdeⅠ, XhoⅠ), the target gene DT388sBAFF was separated by agarose gel electrophoresis, and then inserted into the expression vector pcold II to construct the recombinant expression carrier pcold Ⅱ-DT388sBAFF, transformed into BL21competent fermentation, then positive strains were identified by colony PCR.3. Picked monoclonal colonies were cultured to logarithmic phase, induced by IPTG, the analysis of fusion protein DT388sBAFF through SDS-PAGE and Western blot.4. Ni2+-NTA affinity chromatography fusion protein DT388sBAFF, after dialysis, SDS-PAGE detection of fusion proteins.Results:1. Successfully constructed the recombinant plasmid pMD19-T-DT388sBAFF, the gene sequence cloning purposes sequencing results DT388sBAFF gene sequences exactly.2. Recombinant expression vector pcold II-DT388sBAFF transformed BL21, induced by IPTG ultrasound sonicated by SDS-PAGE results showed that the inclusion body58.4KD at apparent protein bands, in line with the target protein size, image scanning analysis showed that obtained purposes protein of total bacterial protein40%, the results of Western blot show fusion protein DT388sBAFF with anti-His-Tag polyclonal antibody and anti-BAFF polyclonal antibody could specifically reacted, that get the fusion protein-specific fusion protein DT388sBAFF and form of inclusion bodies.3. Fusion protein DT388sBAFF by Ni2+-NTA column purification, scanning gel image analysis, the fusion protein DT388sBAFF more than90%purity.Conclusions:The use of cloning technology to successfully build a diphtheria toxin/human B cell-activating factor (DT388sBAFF) prokaryotic expression plasmid pcold Ⅱ-DT388sBAFF of the state strains stably expressing the engineered strain was transformed into BL21feelings; explore the optimized recombinant protein DT388sBAFF expression conditions, bear with meextraction step and the purification process. PART2Detection biological activity of Diphtheria toxin/human B-cell activating factor fusion proteinBackground:BAFF (B lymphocyte activating factor), also known as BLyS(B-lymphocyte stimulator), TALL-1(TNF-and APOL-related leukocyte expressed ligand1), or THANK(TNF homolog that activates apoptosis, NKFB, and JNK), is a member of TNF (the tumor necrosis factor) superfamily [5-6] and is expressed on the surface of monocytes, DC (dendritic cells), neutrophils, stromal cells, activated T cells, malignant B cells and epithelial cells[7-8]. BAFF binds to three different receptors, BAFF-R, TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) and BCMA (B cell maturation protein), which are expressed differentially at various times during B cell ontogeny [9]. BAFF-R specifically expresses on more mature B cells, starting at the T1transitional B cell stage [7]. Nevertheless, a recent research [9] presented evidence that the BAFF-R is universally expressed on B-lineage acute lymphoblastic leukemia (ALL) cells which develop by transformation of normal B-cell progenitors. And other studies have shown that the fusion protein constructed by BAFF and Gelonin, a plant toxin, demonstrated killing effects against BAFF-R (+) B lymphoma cells.The killing effect of the fusion protein the BAFF-R (+) B cell lymphoma cells depends on two aspects, the first is the binding ability fusion protein with such cells, the other is the killing effect of a fusion protein toxic group to such cells.Binding ability primarily by the expression of cell surface the BAFF receptor capacity decided. Hmy2.CIR cells using B-lymphoblastoid cell constructed experimental cell lines. Real-time fluorescence quantitative the PCR technique Hmy2.CIR cells on BAFF receptor mRNA were detected using fluorescein-labeled the recombinant immunotoxin Hmy2.CIR cell binding capacity last cytotoxicity assay fusion protein for Hmy2.CIR cells killing effect.Purpose:To detect the biological activity of Diphtheria toxin/human B-cell activating factor fusion protein. Methods:1.Using real-time quantitative PCR detection of B-cell lines Hmy2.CIR cells on BAFF three receptors (BAFF-R, TACI and BCMA) expression levels.2.Using FITC fluorescein-labeled detection the the recombinant immunotoxin DT388sBAFF B cell lines Hmy2.CIR cell binding capacity.3.Cell toxicity assay the recombinant immunotoxin DT388sBAFF killing B cell lines Hmy2.CIR cells.4. Through the function analysis of variance of SPSS18.0statistical software the the results were statistically analyzed.Results:1.The three receptors1.BAFF the BAFF-R, TACI and BCMA B cell lines Hmy2.CIR cells expressed the highest expression of BAFF-R.2. Under a fluorescence microscope FITC-labeled recombinant luciferase the protein DT388sBAFF after inverted to Hmy2.CIR cells, strong green fluorescence can be observed, negative control group U937cells no fluorescent, indicating that the fusion protein Hmy2.CIR cell surface by having a specific binding capability.3.Cytotoxicity assay detection result display the fusion protein DT388sBAFF Hmy2.CIR cells, has a strong inhibitory effect, and the inhibitory effects of a dose-dependent relationship, i.e. with increasing concentrations of the inhibitory effect of the fusion protein, the stronger (P<0.05), while different concentrations of fusion protein inhibitory effect of U937cells of the negative control group was no significant difference (P>0.05).Conclusions:Has laid the foundation for targeted B-cell activity the immunotoxin DT388sBAFF, its B-cell malignancies and autoimmune diseases. PART3The killing effect of Diphtheria toxin/human B-cell activating factor fusion protein to human acute lymphoblastic leukemia BALL-1cellsBackground:Acute leukemia (acute leukemia) is a common childhood tumor blood system, acute lymphoblastic leukemia accounts for about70%, of which80%comes from the B-cell lines showed an upward trend in the incidence and mortality, serious threat to humans, especially children health. The treatment methods commonly used in combination with chemotherapy, but the anti-tumor capabilities of these therapeutic measures, that is, poor selectivity, and have a significant systemic toxicity. Long-term use of non-specific immunosuppressants easily induce drug resistance, and immune function in patients can be severely damaged, leading to many complications.Immunotoxin as a new type of treatment are mainly two characteristics superior to other treatment options:One is the immunotoxin killing of tumor cells does not depend on the host’s immune system, but relies on the targeting vector carrying toxins; Second immunotoxin no killing effect on normal cells, but can be effective within the ingestion of target cells with the characteristics of the specific killing of target cells. Therefore, the use of immunotoxins treatment of cancer has prominent advantages, representing a second revolution in antibody-mediated cancer therapy.Diphtheria toxin fragment C the ribosomal peptide chain can EF-2inactivation, thereby inhibiting protein synthesis, cell degeneration and necrosis, the application of this feature is the role of anti-tumor cell. In this study, by constructing immunotoxins DT388sBAFF to investigate whether human acute leukemia cells BALL-1inhibition, and thus provides a new research direction for the treatment of acute leukemia.Purpose:To study the killing effect of the immunotoxin DT388sBAFF (diphtheria toxin/B cell-activating factor fusion protein) on human acute leukemia BALL-1cell.Methods:1.Using real-time quantitative PCR in human acute leukemia BALL-1cells on BAFF expression levels of three receptors (BAFF-R, TACI and BCMA). 2.FITC fluorescein-labeled detection the recombinant immunotoxin DT388sBAFF acute leukemia BALL-1cells binding capacity.3.The cytotoxicity experimental detection the recombinant immunotoxin DT388sBAFF of the human acute leukemia BALL-1cell killing effect.4. Through the function analysis of variance of SPSS18.0statistical software the the results were statistically analyzed.Results:1.BAFF three receptor (BAFF-R, TACI and BCMA), only BAFF-R in human acute leukemia BALL-1cells, high expression (P<0.05), TACI and BCMA basic non expression (P>0.05).2.FITC fluorescein labeled recombinant immunotoxin DT388sBAFF observed in inverted fluorescence microscope servant acute leukemia BALL-1cells and the positive control Hmy2.CIR cells have a strong green fluorescence, while the negative control group U937cells no fluorescent. And the the acute leukemia BALL-1cells and positive control Hmy2.CIR cells only express both BAFF a receptor:BAFF-R, this indicates that the recombinant immunotoxin DT388sBAFF BAFF-R with said cell surface receptor having a specific binding capacity.3. Cytotoxicity assay test results show of recombinant immunotoxin DT388sBAFF on human acute leukemia BALL-1cells has a strong inhibitory effect, and the inhibitory effects of a dose-dependent relationship, i.e., with the stronger the inhibitory effect of increasing concentrations of fusion protein (P<0.05), this positive control results of cell Hmy2.CIR cell line (P<0.05), and the inhibitory effect to the negative control group U937cells under different concentrations of fusion protein was no significant difference (P>0.05).Conclusions:Immunotoxin DT388sBAFF has targeted killing of human acute leukemia activity of BALL-1cells. This study human provides a new research direction for acute leukemia treatment.