Expression Of Fusion Protein Of Human Soluble B Lymphocyte Stimulator And Diphtheria Toxin In E.coli And Its Biological Activities

Immunotoxin (Immunotoxins, IT), also known as biological missile, is a class of fusion protein designed to destroy the specific cell selectively, which is made up of the highly specific monoclonal antibodies and the lethal toxin by chemical cross-linking. Immunotoxin plays a role in inhibiting protein synthesis after binding receptor of tumor cells directly and internalization, which leading to target cell death. Immunotoxin has effects of not only specific recognition, but also killing function, which overcome the defect of chemoradiation of cancer with the traditional treatment.B lymphocyte stimulating factor(BAFF) is a new member of tumor necrosis factor family, which plays an important role in B cell development and autoimmune disease. BAFF exerts its biological effects by binding to its receptor and participating proliferation and functional regulation of T, B lymphocyte. The lack of BAFF may lead to immunocompromised body, but overexpression of which is closely related with the occurrence and development of a variety of autoimmune diseases.ObjectiveTo explore the effect of human sBAFF-DT388 in treatment of B cell malignancies and autoimmune disease, we conducted the hsBAFF-DT388 fusion protein expression vector, had the recombinant protein purified and made the experimental study of biological activity.Methods1. The hsBAFF-DT388 gene was cloned successfully according to optimized primers, which was inserted into the pMD19-T cloning vector. The positive clone was identified by colony PCR, digestion of restriction enzyme and DNA sequencing. 2. The recombinant cloning vector was digested by incision enzyme Ndeâ… and Xhoâ… . The target gene was separated by agarose gel electrophoresis and inserted into the pColdâ…¡expression vector, and transformed into BL21 competent strains. The recombinant expression vector was identified by colony PCR.3.The monoclonal colony was picked and cultured to logarithmic phase, and then induced by IPTG. The recombinant protein was analysised by SDS-PAGE and Western blot.4. The recombinant protein was purified by Ni2+-NTA affinity chromatography, and then dialysised, detected by SDS-PAGE.5. The recombinant protein was labeled by PE-fluorescence and detected the ability of binding with the receptor.6. It was confirmed that the recombinant protein have cytotoxic effect for BAFF-R(+) B cells (Hmy2.CIR cells) by cytotoxicity assay.SPSS16.0 statistical software used for statistical analysis.Results1. The hsBAFF-DT388 gene was cloned, the recombinant plasmid pMD-hsBAFF-DT388 was successfully constructed. Sequence analysis showed that the cloned gene was same as the optimal sequence of hsBAFF-DT388 gene.2. The positive recombinant plasmid was transformed into competent strain BL21, which was induced by IPTG. The lines of SDS-PAGE analysis showed that there was a distinct protein band in the 58.4KD, which was consistent with the size of the target protein.The analysis of image scanners showed that target protein accounted about 40% of total protein. Western blot showed that recombinant protein can specificly react with both the anti-BAFF polyclonal antibody and anti-His-Tag polyclonal antibody, which indicating that the specificity of the recombinant protein hsBAFF-DT388.3. The gel image analysis of the recombinant protein after purified by Ni2+-NTA affinity chromatography show that the purity of hsBAFF-DT388 recombinant protein account to more than 90%.4. Strong red fluorescent can be observed under the inverted fluorescence microscope when BAFF-R(+) Hmy2.CIR incubated with the recombinant protein labeling fluorescein, while the negative control group BAFF-R(-) U937 cells didn't appear fluorescence. It shows that the recombinant protein has a specific binding ability to receptor of BAFF-R(+) Hmy2.CIR cell.5. It was confirmed that the recombinant protein have cytotoxic effect for BAFF-R(+) B cells (Hmy2.CIR cells) by cytotoxicity assay, which showed a strong inhibitory effect of the recombinant protein, and the inhibitory effect was dependent with dose: the stronger inhibitory effect appears with the increasing concentration of recombinant protein(P <0.05). But the inhibitory effect of different concentrations of recombinant protein in the negative control group BAFF-R(-)U937 cells appears no significant difference(P> 0.05).ConclusionThe hsBAFF-DT388 gene was cloned by PCR amplification, and sequence analysis showed that the cloned gene was same as the optimal sequence of hsBAFF-DT388 gene. The prokaryotic expression vector of the recombinant plasmid pcoldâ…¡-hsBAFF-DT388 was successfully constructed, and transformed into competent strains BL21. Engineering Strains expressing stably was obtained; We explored expression conditions of recombinant pcoldâ…¡-hsBAFF-DT388, and optimized extraction of inclusion bodies and purification of protein; We obtain the recombinant protein with selective cytotoxicity against B cell, which established a solid foundation for further study for the therapy of the B cell malignancies and autoimmune diseases.