Study on allelic characteristics of "homozygote" resulted from low resolution genotyping of HLA-Cw locus / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the allelic characteristics of "homozygote" resulted from low resolution genotyping of HLA-Cw locus and to provide more precise typing data for clinical transplantation.</p><p><b>METHODS</b>Forty three related allogeneic hematopoietic stem cell transplantation(allo-HSCT) donors and patients with HLA-Cw * 03, Cw * 07 homozygote, which were the most common gene groups in Chinese population, identified by low resolution genotyping level were retyped by high resolution PCR-SSP genotyping method, and three dimensional structure modelling was also made by using a solely developed HLA three-dimensional matching software (HLA strucMark version 1.0) to evaluate the effect of differences between two mismatched alleles and its relationship with GVHD occurrence.</p><p><b>RESULTS</b>The typing results of high resolution level demonstrated that the confirmed allelic homozygotes for Cw * 03 and Cw * 07 were 14% and 43%, respectively, while the others were all heterozygotes. The ambiguous typing results could be confirmed by family data study or by high resolution typing methods when there was no family data available, Three-dimensional modeling results of the mismatched alleles undetected in low resolution typing level indicated that family data study or high resolution PCR-SSP genotyping were important in preventing graft-versus-host disease.</p><p><b>CONCLUSIONS</b>When HLA-Cw homozygote results were found in low resolution genotyping method, the results should be reconfirmed by family data study or by high resolution typing methods to provide precise typing results for avoiding severe graft-versus-host disease.</p>

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.

Haplotype and linkage analysis of HLA-I classical genes in Chinese Han population / 中国实验血液学杂志

The aim of this study was to investigate the parameters of gene frequencies, haplotype frequencies and linkage disequilibrium of HLA-A, -B, -Cw in HLA classical I loci for Chinese Han population. HLA-A, HLA-B and HLA-Cw loci were genotyped in 1014 unrelated China people using low resolution PCR-SSP typing method, and their genetic parameters were analyzed by statistic methods. The results indicated that among all the detected HLA-I genes, A*02 (0.33), A*11 (0.24), B*15 (0.14), B*13 (0.13), Cw*03 (0.25) and Cw*07 (0.18) were the popular gene groups distributing in Chinese Han population, and A*02-B*46 (0.071), A*11-B*15 (0.051), A*02-Cw*01 (0.084), A*11-Cw*03 (0.079), B*46-Cw*01 (0.095) and B*13-Cw*03 (0.071) were the predominant haplotypes in Han population. Additionally, A*02-B*46, A*30-B*13, A*30-Cw*06, A*02-Cw*01, B*46-Cw*01 and B*58-Cw*03 were statistically significant with strong linkage disequilibrium. While A*02-B*15, A*02-B*40, A*24-Cw*03, A*02-Cw*03 and A*31-Cw*03 were in low linkage disequilibrium, among them A*24-Cw*03 appeared frequently in HLA recombination events. In addition, A*02-B*46-Cw*01 (0.075), A*30-B*13-Cw*06 (0.046), A*11-B*13-Cw*03 (0.045), A*33-B*58-Cw*03 (0.044), A*11-B*15-Cw*08 (0.027), A*02-B*38-Cw*07 (0.023) and A*11-B*40-Cw*07 (0.022) were the main extended haplotypes in Han population. In conclusions, this study investigated systematically the genetic polymorphism features of Chinese Han population, which may provide useful genetic parameters for researches in colonial evolution, clinical transplantation and disease susceptibility.

Genetic polymorphism of HLA-Cw locus in hematopoietic stem cell transplantation patient and donor population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the distribution of genes in HLA-Cw locus from Han population of China in a large scale, and to provide basic data for further study on the genetic characteristics of HLA-Cw locus of this population.</p><p><b>METHODS</b>Totally 1285 unrelated Chinese Han individuals were typed by PCR-SSP, and statistics was utilized to investigate the distribution rules of detected genes.</p><p><b>RESULTS</b>Twenty-three HLA-Cw alleles were identified in Chinese Han population, out of them HLA-Cw*01, *03, *07 and *08 were the commonest genes, which accounted for frequencies of 0.1529, 0.2385, 0.1747 and 0.1004, respectively. Five genes which could not be identified by serological method were deaed: HLA-Cw*12, *14, *15, *16 and *17. Hardy-Weinberg test showed that the observed genetic polymorphism distribution values were correspondent with the expected (chi-square=73.74, df=98, P>0.5).</p><p><b>CONCLUSION</b>This study may serve a full-scale scientific genetic parameters of HLA-Cw genes for Chinese Han population studies.</p>

Establishment of purification procedure for recombinant fusion protein B7-2-PE40KDEL / 中国实验血液学杂志

This study was aimed to establish downstream purification procedure by which the protein of interest can be purified to higher purity rapidly and efficiently. The different combinations of various purification strategies, methods and conditions were compared, including reversed phase chromatography, metal chelating chromatography, anion exchange chromatography, blue dye affinity chromatography, filtration chromatography and so on. The results showed that in reversed phase chromatography, isolated protein of interest was denatured and precipitated immediately after chromatography because methanol or acetonitrile were adopted as the organic phase. In blue dye affinity chromatography expecting to purify the protein of interest in one step, protein of interest was difficultly differentiated from mixed protein as much proteins bound to the chromatography media by non-specific affinity. While there is a translation-enhancing sequence T7-g10 in the PRSETA-B7-2-PE40KDEL expression vector, so it adds 6 histidines to the N terminus of the protein of interest, this allows to purify the protein of interest by metal chelating chromatography. Based on this characteristic, a three-step chromatography line including metal chelating chromatography, anion exchange chromatography and filtration chromatography was finally established after repeated experiments. By this way the purity of protein of interest reached 95% and the total recovery rate was 8%. The result of Western blot indicated that the expressed and purified recombinant B7-2-PE40KDEL could specifically bind with mAb against human B7-2 and multiple antibody against PEA. The cytotoxicity of the recombinant toxin tested by MTT method showed that the B7-2-PE40KDEL could selectively kill Jurkat cell line expressing CD28 receptor well and had no killing effect on the Raji cell line unexpressing CD28 receptor. It is concluded that a high efficient and speedy three-step purification procedure for the purifying recombinant protein B7-2-PE40KDEL was established, and this procedure possess selective killing activity on CD28 positive T lymphocytes.

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>

Recombination within the human leucocyte antigen / 中华医学遗传学杂志

Understanding with greater clearness the characteristics of recombination within the human leucocyte antigen(HLA) is of deep significance to gaining an insight into the evolutionary process of shaping HLA allelic diversity and ultimately the human resistance against diverse pathogens. Family studies and statistical analysis of recombination have provided estimations of recombination fractions across the major histocompatibility complex and have identified the potential recombination hotspots. Other characteristics such as haplotype specificity and sequence motifs have been intensively studied. The recombination fractions, hotspots and other characters are reviewed in this paper.

Fusion construction, prokaryotic expression and structure characteristics prediction of bimolecular thrombopoietin / 中国应用生理学杂志

<p><b>AIM</b>Design, construction, expression in E coli and protein characteristics prediction of bimolecular thrombopoietin (T-T) with more stability, efficiency, and lower toxicity.</p><p><b>METHODS</b>The expression vectors of TPO and T-T, pET32 a(+)/TPO and pET32 a (+)/T-T, had been constructed by molecular cloning methods. Then, they were expressed in host bacterium. Their products were identified by Western blot. The protein characteristics, such as second structure, antigenicity, hydrophilicity, flexibility and isoelectric point, were predicted by DS Gene and Protscale software.</p><p><b>RESULTS</b>The expressing vectors pET32a(+)/TPO and T-T were constituted correctly and expressed in origami (DE3), and their expression efficiency were more than 40 percent of total protein. T-T was identified correctly by Western blot. DS Gene and Protscale software predict the protein characteristics of TPO sequences in T-T molecule were no change, there was high flexibility in the linker domain. But two amino acids in T-T molecule have been mutated, and an insert fragment with 34 amino acids following the linker had antigenicity, hydrophilicity, and beta-sheet structure.</p><p><b>CONCLUSION</b>We have constructed correctly and expressed T-T with high level in E Coli. Protein characteristics prediction of T-T accords with our design.</p>

Biochemical and physical properties for a recombinant IL6 Pseudomonas exotoxin fusion protein IL6D24-PE40KDEL / 中国实验血液学杂志

The objective was to identify some biochemical and physical properties for fusion protein IL6D24-PE40KDEL. Edman degradation, SDS-PAGE, peptide mass fingerprinting, Western blot and MTT were used for identification of the protein. The results showed that the sequence of N-terminus is Met-Ile-Asp-Lys-Gln-Ile, Met was added because of prokaryotic expression system; Western blot revealed that the purified protein could react with IL6 and PEA antibody. The purified protein IL6D24-PE40KDEL could kill the multiple myeloma cell lines U266 expressing high affinity IL6R, but it could not kill the cell lines CEM which not expressed IL6R; The molecular weight was 58.7 kD measuring by SDS-PAGE; peptide mass fingerprinting (PMF) confirmed that the construction of IL6D24-PE40KDEL was correct. A novel protein by Peptident database in EXPASY web site was identified. In conclusion, IL6D24-PE40KDEL is a new targeting protein with bioactivity of specific killing effect.

Effects of cryopreservation length on the proliferative potential of cord blood hematopoietic cells in vitro / 中国应用生理学杂志

<p><b>AIM</b>To study the effects of cryopreservation length on the proliferative potential of hematopoietic cells derived from cord blood.</p><p><b>METHODS</b>Using Dextran-40 and 10% DMSO as cryoprotectants, separated nuclear cells were stored in liquid nitrogen after they were freezed according programme. One month or 4 months later, they were thawed and expanded in serum-free medium for culture and expansion of hematopoietic cell (SFEM) for 5 weeks. Dynamic results were detected every week.</p><p><b>RESULTS</b>At the 5th week of expanding, TNC were expanded for 1499.0 +/- 115.6-folds and 1513.0 +/- 110.4-folds, respectively. CD34+ cells and CFCs reached to their highest level at the 2nd week and at the 3rd week. CD34+ cells were expanded for 63.8 +/- 6.1-folds and 62.4 +/- 5.7-folds, respectively. CFCs were expanded for 53.8 +/- 6.3-folds and 54.8 +/- 6.7-folds, respectively. Between the two kinds of cells, statistical significant difference in proliferative potential wasn't detected.</p><p><b>CONCLUSION</b>In ideal cryopreservative condition, the cryopreservation length would do not affect the proliferative potential of cord blood hematopoietic cells.</p>

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 probability of microbiological contamination during the collection and processing of umbilical cord blood / 中国实验血液学杂志

To study the pathogens incidences in cord blood and the efficiency of different detective methods, 60 samples were drawn and reserved from collected and processed cord blood, respectively. The BACTEC 9050 system, improved Martin/thioglycollate broth (22 degrees C) and thioglycollate broth (35 degrees C) were employed to detected bacteria (including fungus) at the same time. Two hundred and six cord blood serum samples were used to detect the HBV DNA and HCV RNA by molecular biology technique, HBsAg, Anti-HBC, Anti-HCV, Anti-HCMV-IgM, HTLV-1, HTLV-2, HIV-1 and HIV-2 by ELISA and RBC agglutination test were used to detect the TPHA. Results showed that using BACTEC 9050 system, the incidence of bacteria and fungus was 3.33% and 0% respectively in collected cord blood; in processed cord blood, the rates increased to 6.67% and 1.67%, respectively. The sensitivity of BACTEC 9050 was higher than that of Martin/thioglycollate broth (22 degrees C/35 degrees C) culture. In 206 serum samples, the positive rate of HBV DNA was 5.8%, HCV RNA was 2.4%, HBsAg was 2.4%, HCMV-IgM was 1.89%, HCV was 2.4% and Anti-HBC was 29.4%. In those samples that Anti-HBC was positive, the positive rate of HBV DNA was 6.7%. It was concluded that the incidences of microbiological contamination in cord blood were high. The routine culture system would lead to false negative results of obligate anaerobes. It was necessary to replace the current culture system with improved system, such as BACTEC 9050 system. The molecular biology technique would make up for the default of ELISA.

A Strategy of IL-6/IL-6R System Mediates Targeted Treatment for Leukemia / 中国实验血液学杂志

An inevitable trend for the development of new treatment of leukemia is to use targeted strategy. IL-6/IL-6R is important one of the cytokine receptor targeted treatment systems. Many malignant cells, including multiple myeloma, prostate carcinoma and leukemia etc., have been shown to express IL-6R. Leukemia cells, especialy in U937, TF1, KG1 cell lines highly express the high affinity IL-6R. IL-6 recombinant toxin is cytotoxic in vitro to leukemia cell expressing high affinity IL-6R; in vivo the fusion toxin can result the reduction of leukemic cell load in animal leukemia model but have no effect on normal hematopoiesis in non-leukemic animal. On the basis of these pre-clinical studies, IL-6 recombinant toxin my become novel drug for the treatment of leukemia and cancer in future.

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.