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Diphtheria Toxin/Human B-Cell Activating Factor Fusion Protein Kills Human Acute Lymphoblastic Leukemia BALL-1 Cells: An Experimental Study

Diphtheria Toxin/Human B-Cell Activating Factor Fusion Protein Kills Human Acute Lymphoblastic Leukemia BALL-1 Cells: An Experimental Study
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摘要 Objective: This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor (DT388sBAFF) in Escherichia coli (E. coli) and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1). Methods: A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction (PCR) and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and then purified by Ni2+-NTA affinity chromatography. The expression level of B cell-activating factor receptor (BAFF-R) on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion: The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia. Objective: This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor (DT388sBAFF) in Escherichia coli (E. coli) and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1). Methods: A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction (PCR) and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and then purified by Ni2+-NTA affinity chromatography. The expression level of B cell-activating factor receptor (BAFF-R) on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion: The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.
出处 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 2012年第3期238-244,共7页 中国癌症研究(英文版)
基金 supported by grants from the National "973" Basic Research Program of China (No.2012CB944703) the National Key Technology Research and Development Program of China (No.2011BAI17B00) the Shandong Provincial Science and Technology Development Projects (No.2009GG10002008 and No.2011GSF12103)
关键词 B cell-activating factor B-lineage acute lymphoblastic leukemia Diphtheria toxin Fusion protein B cell-activating factor B-lineage acute lymphoblastic leukemia Diphtheria toxin Fusion protein
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参考文献22

  • 1Kreitman RJ. Recombinant immunotoxins containing truncated bacterial toxins for the treatment of hematologic malignancies. Bio Drugs 2009; 23:1-13.
  • 2Kreitman RJ. Immunotoxins for targeted cancer therapy. AAPS J 2006; 8:Es32-s1.
  • 3Lyu MA, Cao YJ, Mohamedali KA, et al. Cell-targeting fusion constructs containing recombinant gelonin. Methods Enzymol2012; 502:167-214.
  • 4FitzGerald DJ, Wayne AS, Kreitman RJ, et al. Treatment of hematologic malignancies with immunotoxins and antibody-drug conjugates. Cancer Res 2011; 71:6300-9.
  • 5Zhang J, Roschke V, Baker KP, et al. Cutting edge: a role for B lymphocyte stimulator in systemic lupus erythematosus. J Immunol 2001; 166:6-10.
  • 6Zhou T, Zhang J, Carter R, et al. BLyS and B cell autoimmunity. Curr Dir Autoimmun 2003; 6:21-37.
  • 7Mackay F, Silveira PA, Brink R. B cells and the BAFF/APRIL axis: fast?forward on autoimmunity and signaling. Curr Opin Immunol 2007; 19:327-36.
  • 8Liu Z, Davidson A. BAFF and selection of auto reactive B celis. Trends Immunol 2011; 32:388-94.
  • 9Bossen C, Schneider P. BAFF, APRIL and their receptors: structure, function and signaling. Semin Immunol 2006; 18:263-75.
  • 10Ng LG, Sutherland AP, Newton R, et al. B cell-activating factor belonging to the TNF family (BAFF)-R is the principal BAFF receptor facilitating BAFF costimulation of circulating T and B celis. J Immunol 2004; 173: 807-17.

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