Prokaryotic expression of HLA-B * 2705 heavy chain and identification for its activity / 中国实验血液学杂志

In order to investigate the expression of heavy chain of HLA-B * 2705 in prokaryotic system and identify its activity, the extra-membrane gene fragment of HLA-B * 2705 was amplified from full-length HLA-B*2705 cDNA by PCR and cloned into pGEM-T vector. After identification by sequencing, the prokaryotic expressing vector pET32a (+)-B * 2705 was constructed. The antigenic activity of expressed protein was identified by Western blot and antibody blocking reaction. The results indicated that the fused HLA-B * 2705 protein expression with high efficiency was obtained. The expressed product was more than 50% of the total bacteria protein. The antigenic activity of expressed protein was confirmed by Western blot and antibody blocking reaction. It is concluded that HLA-B * 2705 fusion protein are obtained as basis for the further studies.

A preliminary study on the expression and biological function of recombinant human SCF-TPO fusion protein / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expression of recombinant human SCF-TPO fusion protein and its biological function.</p><p><b>METHODS</b>Four primers were designed according to known sequences of TPO and SCF. The functional amino acid domains of TPO and SCF were amplified by RT-PCR from fetus hepatocytes, respectively. The expression plasmid pET32a/SCF-TPO was constructed by VOE gene fusion technique and expressed in E. coli BL21(DE3) plysS as inclusion body after isopropyl-beta-D-1-thiogalactopyranoside (IPTG) induction. The fusion protein was tested by SDS-PAGE and Western blot. The biological functions of SCF-TPO fusion protein in MO7e cells was investigated by MTT method after purification with metal chelating chromatography.</p><p><b>RESULTS</b>The high expression SCF-TPO fusion protein was obtained, reaching up to 30% of the total cellular protein. Western blot verified the correct fusion expression and MTT results showed the growth promoting effect of the SCF-TPO fusion protein on MO7e cells, with a higher promoting activity at 100 ng/ml.</p><p><b>CONCLUSIONS</b>Expressed SCF-TPO fusion protein after renaturation has biological activity in promoting the proliferation of MO7e cells.</p>

Prediction of acute GVHD after umbilical cord blood transplantation by HLA three-dimensional structure matching / 中国实验血液学杂志

To explore a method of predicting acute graft versus host disease (aGVHD) after unrelated cord blood transplantation (UCBT), the HLA-A, -B, -DRB1 molecular three-dimensional structures in 25 patients with blood disorder who underwent UCBT and their donors were modeled by using molecular modeling technique. First, full amino acid sequences of each HLA antigen from HLA data banks were loaded down, and then amino acid sequence of extracellular antigen binding region was chosen. Third step, SPDBV software of SWISS-MODEL server was used to modeling the three-dimensional structures of each different allele of HLA-A, -B and -DRB1 between patients and donors and the parameter "root mean square deviation" (RMSD) was used to indicate the structure differences. Last, RMSD of each different HLA allele of each donor-patient pair were added together to get total RMSD. The 25 patients were divided into 3 groups: the first group did not develop aGVHD; the second group developed aGVHD graded I-II and the third group developed aGVHD graded III-IV. The results showed that in the 25 patients divided into three groups, 8 patients in the first group did not develop aGVHD (32%); 13 patients in the second group developed grade I-II of aGVHD (52%) and 4 patients in the third group developed aGVHD III-IV (16%). The total RMSDs of each group were 0.24 +/- 0.15, 0.25 +/- 0.14 and 0.47 +/- 0.22 respectively. The total RMSD of the third group was significantly higher than that of the other two groups. In conclusion, utilization of modeling HLA molecular three-dimension can predict the severe aGVHD after UCBT quickly, simply and accurately. It provides scientific basis in choosing a optimal cord blood donor to avoid severe aGVHD for physicians and the cord blood banks. And it is instructive too to direct the application of immunosuppressive agents after transplantation in clinic.

Expression of alpha subunit for IL-6 receptor at mRNA and protein levels in human leukemic cells / 中国实验血液学杂志

To probe into the expression of alpha subunit for IL-6R at both mRNA and protein levels in human leukemic cells and to discuss its implication in targeted treatment for leukemia with recombinant IL-6-PE40 exotoxin fusion protein mediated by IL-6/IL-6R system, semi-quantitative RT-PCR, sequencing and FCM were used to analyze the gene and protein expression levels of alpha subunit for IL-6R in leukemic cells. Our results showed that not only mRNA but also protein of alpha subunit for IL-6R are highly expressed in the myelogenous leukemic cell lines, the relative expression levels of mRNA were KG-1(1.22) > (1.02) > U266(1.00) > U937(0.99) > HL-60(0.76). Among lymphoblastic leukemic cell lines, Raji expressed a certain amount of alpha subunit mRNA (0.77), whereas its alpha subunit protein was not detected. Expression of alpha subunit mRNA and protein were negative in lymphoblastic leukemic cell lines, HuT28 and CEM, and chronic myelocytic leukemic cell line K562. These results correlate with those of FCM highly. Noteworthily, normal human peripheral blood mononuclear cells expressed hardly protein of IL-6R alpha subunit. So this study provides sufficient experimental evidence that the targeted treatment by recombinant IL-6-PE40 can specifically kill leukemic cells highly expressing IL-6R without toxicity to normal hematopoietic cells.

Advances in research on cytokine mimetic peptides/non-peptides stimulating hematopoiesis / 中国实验血液学杂志

Cytokines such as erythropoietin (EPO) and thrombopoitein (TPO) and so on, which stimulate hematopoiesis, can regulate self-renewal, proliferation, differentiation, maturation and programmed cell death of hematopoietic cells through specifically binding to surface receptors. Recently random phage display peptide libraries and other screening methods have been used to isolate mimetic including small peptides and non-peptides molecules, which can mimic the same effects as cytokines, such as EPO and TPO, and demonstrate the similar potency and activity as EPO and TPO in a panel of in vitro biological assays and in animal experiments. These approaches are critical to further research of interactive mechanisms between cytokine and receptor, receptor activation and rational design of other desired cytokine mimetic. This review concisely introduced recent advances in research on mimetic of EPO, TPO and other cytokines and future directions.

Cloning, expression and biological activity identification of a cDNA encoding the extracellular region of human b7-2 / 中国实验血液学杂志

As one important member of B7/CD28/CTLA-4 costimulatory signal pathway, B7-2 molecule plays a critical role in regulating T-cell response. In order to further explore its effects on regulation of T cell activation, proliferation and associated signal pathways, the cDNA encoding extracellular region of human B7-2 was amplified via PCR and subcloned into some prokaryotic expression vectors to express target protein in host strains. The expressed protein was identified with Western blot and MTT. Results showed that after screening, the expression level of the protein of interest attained the yield of over 20% total bacterial protein by using pGEX-4T-2 vector and E. coli BL21 (DE3)-CodonPlus-RIL host cells. The recombinant protein could specially react with B7-2 McAb and could stimulate T-cell proliferation combined with anti-CD3 antibody. In conclusion, the recombinant protein was bioactive, therefore the study will make it possible for the research of relationship between B7-2 structure and its function.

Selection of donor in mismatched hematopoietic stem cell transplantation by CREG, residue match and HLA three-dimensional structure / 中国实验血液学杂志

After search at hematopoietic stem cell banks for transplant donors, there may be several donors matched with given standards. To determine the most appropriate donor for a specific patient, the potential donors were analyzed and compared by three methods. The first is cross-reactive group (CREG) antigens, which defined as the public antigens that shared specific serological reaction patterns. The second is residue match theory, which concerned the three residues oriented upward toward the T-cell receptor. The third is comparing HLA three-dimensional structure models. The results of the three methods were not completely accorded in our case. However, some less matched donors could be excluded from the candidates and the range of selection was further reduced. It is concluded that combined application of three methods would contribute in selecting donor for hematopoietic stem cell transplantation in clinics.

The Prediction of the Molecular Biology Characteristics of Recombinant Fusion Exotoxin B7-1-Linker-PE40 and B7-2-Linker-PE40 / 中国实验血液学杂志

In order to confirm the reasonability of designed recombinant exotoxin B7-1-Linker-PE40 and B7-2-Linker-PE40, their molecular biology characteristics, such as flexibility, antigenicity, hydrophilicity, epitope and secondary structure, were predicted by using a computer software GOLDKEY. It had been found that the recombinant fusion exotoxin had kept the epitope characterstics of B7-1, B7-2 and PE40, and had not got new epitope, and the antigenicity in flexible linker was extxemely low. The linker inserted in the recombinant fusion exotoxin had low epitope, low antigenicity and high flexibility. Compared to B7-1, B7-2 and PE40, there are several amino acid residues changes in B7-1-Linker-PE40 and B7-2-Linker-PE40, respectively, which might have some effect on secondary structure of the recombinant fusion exotoxins. Western blot analysis revealed that both B7-1-Linker-PE40 and B7-2-Linker-PE40 could bind specifically with antibodies against B7-1, B7-2 and PE40, respectively. The result of Western blot was consistant with the computer prediction that the recombinant proteins retain the antigenicity and spacial structure of B7 and PE40. It is suggested that both fusion proteins designed and constructed were resonable and computer analysis would be helpful for us to study the biological activity of the recombinant fusion exotoxin B7-1-Linker-PE40 and B7-2-Linker-PE40 and construct other recombinant proteins further.

A New Criterion for Donor and Recipient Selection in Hematopoietic Stem Cell Transplantation - the Matching of Three-Dimensional Structure of HLA Molecular Modeling / 中国实验血液学杂志

The purpose of the research is to provide a new standard for matching of HLA three-dimensional structure, and summarize the major permissible mismatch and immunogenic mismatch antigens. The molecular modeling method was used to create HLA molecular structures by Swiss Model Server, and the comparison of the differences among the alleles was done by SPDV software with the function of iterative magic fit. The results were recorded by relative mean square deviation (RMSD, nm). The differences among alleles were scattered below 0.06 nm for HLA-A and -B molecules, and below 0.03 nm for HLA-DRB1 molecules. On the basis of the statistical analysis, when RMSD is greater than 0.04 nm for -A and -B molecules and 0.02 nm for -DRB1 molecules, the difference is meaningful and can be related with graft versus host disease. When RMSD is lower than 0.02 nm for -A and -B molecules and 0.01 nm for -DRB1 molecules, the difference is decided unmeaningful. From the data, the permissible mismatch and immunogenic mismatch alleles within HLA-A, HLA-B and HLA-DRB1 molecules were summarized. Three-dimensional structure matching is a new area in the transplantation field, much research should be done in the future.