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.

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.

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.