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

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 ß-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 Ni(2+)-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.

[Cloning and expression of human interleukin-26 in Escherichia coli].

To clone human interleukin-26 (hIL-26) and express it in E. coli efficiently. Two pairs of primers were synthesized according to the hIL-26 gene reported on GenBank. The hIL-26 gene was cloned by nest PCR following the first round RT-PCR from human peripherial blood monocytes total RNA, and then the PCR product was cloned into pMD18-T vector. Colony PCR, restriction analysis and sequence analysis showed that the gene cloned was the same as the reported hIL-26. The recombinant was cut with BamHI and EcoR I to obtain the hIL-26 fragment, and then the fragment was inserted into pBV220 which was cut with the same enzymes. The recombinant expression vector was induced to express hIL-26 at 42 degrees C, SDS-PAGE analysis showed that the recombinant protein accounted for up to 20% of the whole protein of E. coli, and the protein was also confirmed by Western blotting. Purity of the protein was found to be above 90% after purified with molecular sieve. After renaturalized with glutathione buffer, the promoting effect of it on the production of IFN-y in PBMC was detected by RT-PCR. A recombinant bacterial strain for expressing hIL-26 with biological activity was constructed successfully.

[Correlation of thrombosis with increased platelet turnover in essential thrombocythemia].

This study was aimed to investigate the correlation of thrombosis with increased platelet turnover in essential thrombocythemia. According to presence or absence of thrombosis, 26 patients with ET were divided into two groups. Reticulated platelets (RP) were measured by flow cytometry and 26 healthy volunteers were selected as healthy controls. The ET patients with thrombosis were treated with hydroxyurea and interferon-alpha. The results demonstrated that the ET patients with thrombotic events had a significantly higher RP percentage (14.8% +/- 7.2%) than that in both asymptomatic ET patients (4.5% +/- 2.3%) and normal control (3.3% +/- 1.5%), (P < 0.05); the RP percentage in asymptomatic ET patients did not differ significantly from controls. ET patients with thrombosis also had a significantly higher absolute RP (ARP) count than those in ET patients without thrombosis [(176 +/- 37) x 10(9)/L vs (46 +/- 12) x 10(9)/L]. The ET patients with thrombosis were successfully treated with hydroxyurea plus INF-alpha, the RP percentage and ARP counts obviously reduced. In conclusion, when the ET patients had thrombotic events, those patients had a significantly higher RP percentage and ARP compared with patients without thrombosis and healthy controls. The ET patients with thrombosis were successfully treated with hydroxyurea plus INF-alpha.