| CD26 is a typeâ…¡transmembrane glycoprotein, which is highly presented in various cells. Its DNA sequence and three-dimensional structure have been clearly identified. It is known as dipeptide peptidaseâ…£(DPPâ…£), to be a proteolytic enzyme. The natural substrates of CD26 have many different vareties and exert important functions in vivo. As a receptor and costimulatory protein, CD26 is involved in cell adhesion and apoptosis and plays a major role in immunoregulation and cell migration in many diseases. Inhibitiors of CD26 may be used treat autoimmune and cell proliferative diseases.CD26 is a dipeptidase that preferentially cleaves peptides from the N-terminus with the sequence"Gly-Pro-Xaa"or"Ala-Pro-Xaa"in the penultimate position. Stromal cell-derived factor 1(SDF-1) is the natural substrate of the CD26. SDF-1 and its receptor play an important role in some physiological and pathological processes, such as cell migration, leukocytic infiltration, and allelotaxy. Christopherson reported G-CSF and GM-CSF induced upregulation of CD26 peptidase inhibiting the functional chemotactic response of CD34+CD38- human cord blood hematopoietic cells. Its mechanism is that CD26 can truncate SDF-1, so the CD34+ haemopoietic stem cells (HSCs) degrade the capacity to home to the bone marrow, and inhibition of CD26 may enhance this capacity of HSCs.Our previous research found that haemopoietic stem cells transplanted in human-to-swine hematopoietic chimera may stop in immature stage when they differentiate to erythron because of immunologic rejection by swine. It indicates that transplanted HSCs have not been homing and engrafted to bone marrow successfully, or the rate of homing is low. In order to enhance the functional chemotactic response of HSCs by CD26, in this study, we tried to use EPO to stimulate the efficacy of CD26 on the CD34+ human cord blood hematopoietic cells in vitro, and prepare specific inhibitive monoclonal antibody against the catalytic center of CD26.ObjectiveTo observe the effect of CD26 on the CD34+ human cord blood hematopoietic cells by short-term EPO treatment in vitro. To construct the prokaryotic expression vector with CD26 catalytic center genes, express it highly in E.coli to get purified CD26 fusion protein. To prepare monoclonal antibody against CD26 catalytic center by hybridoma technique and identify it.Methods1. The CD34+ cells were sorted from human cord blood by magnetic activated cell sorting (MACS) technique, and subjected to EPO treatment. We detected CD26 expression by flow cytometer and CD26 activity by responding with chromogenic CD26 substrate Gly-Pro-p-nitoanilide.2. To construct prokaryotic expressive vector and express CD26 fusion protein. The coding gene of CD26 amplified using RT-PCR technology with mRNA template from human leucocyte was inserted into prokaryotic expressive vector PET-32a, and then the recombinant plasmids were transformed into BL21 and induced by IPTG. The fusion protein was purified by Ni+ affinity column chromatography and detected by Western Blot analysis.3. To prepare monoclonal antibody against the catalytic center of CD26. BALB/C mice were immunized with his-CD26 fusion protein and monoclonal antibodies against CD26 were prepared with hybridoma method. The positive hybridomas secreting anti-CD26 McAb were screened by means ELISA. The McAb specificity was detected by Western Blot analysis and immunocytochemistry. The immunoglobulin (Ig) subtype, the titer and the epitope of the McAb were determined by ELISA test, and the chromosomes of the McAb were analyzed as well.Results1. To test the percentage of CD34+ cells treated by EPO after 18h. Culturing of cells in the absence or presence of EPO resulted in no significant change in CD34+ cell numbers. However the addition of 100U/ml or 200U/ml EPO resulted in statistically significant increase in the percentage of CD26+ cells within the CD34+ cell population. The percentage of CD26+ cells within the CD34+ cell population has a rising tendency with the increase of the EPO concentrations. With the increase of the EPO concentrations, the levels of CD26 activity on the surface of CD34+ cells may increase, especially increase significantly after EPO 100U(P<0.05).2. The target gene segment about 660bp, just the same size as expected, was amplified by PCR. After double enzyme digestion identification and sequence analysis, the recombinant plasmids were successfully cloned. And through successful transformation and induction, we got the expression molecular weight 41KDa just the same as expected. Lysated with supersonic, the fusion protein formed inclusion bodies in prokaryotic expression system. The inclusion bodies were isolated from the E.coli and dissolved in denature agent. The proteins have gotten the purity to immunize BALB/C mice after purified by Ni2+ chelating affinity chromatography. The Western Blot analysis showed that inductive expressed fusion proteins could have specific bindings with CD26 goat anti-human polyclonal antibody (R&D), and the band was clear.3. From over thirty positive hybridomas which secreting monoclonal antibody to CD26 fusion protein, we screened out 4 hybridomas, named 1-F3, 1-F1, 3-C3 and 1-F6. Chromosome analysis revealed that the selected hybridoma was with the universal characteristics of the monoclonal hybridoma cells which secreted McAb. The Ig subtypes of them are IgG1, IgG1, IgG1 and IgG3 subclass respectively. BALB/C mice were primed with pristine and then inoculated with well-grown hybridomas intraperitoneally. The Western Blot detection showed that McAbs could bind specifically to the CD26. By immunocytochemistry testing, these 4 McAbs reacted with H9 cell line which expresses CD26 positively. The Western Blot analysis in recognizing epotope of McAbs showed that 1-F3 specifically binds to 710aa-766aa in CD26 gene order, and the other three McAbs specifically binds to 552-632aa.ConclusionsIn our research, the percentage of CD26+ cells within the CD34+ cell population cultured with EPO100U/ml or EPO200U/ml 18h increased significantly compared with the cultured without EPO. In addition, CD26 activity of CD34+ cell population cultured with EPO100U/ml or EPO200U/ml 18h increased significantly also. Short-term EPO treatment in vitro upregulates CD26 expression and activity on the CD34+ human cord blood hematopoietic cells as well as enhances its biological function. And then, the CD26 fusion proteins about 41KDa were obtained by recombinant DNA techniques and prokaryotic expression methods, and the specific monoclonal antibodies against the catalytic center of CD26 were prepared. The obtained monoclonal antibodies with high biological activity belong to the subtypes IgG1, IgG1, IgG1 and IgG3, which served as the foundation for further research on biological functions of inhibitional McAb and enhancement the efficency of homing and planting of HSCs.
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