Sequence analysis of exons 1, 2, 3, 4 and 5 of the HLA-B5/35 cross-reacting group

The HLA-B5/35 cross-reacting group (CREG) is a set of closely related antigens including HLA-B35, B51, B52, B53 and B78. The nucleotide sequences of exon 1 through 5 of the B5/35 CREG were determined to assess the level of polymorphism. For exons 2 and 3, the previously described sequence-based typing (SBT) strategy was applied, the nucleotide sequences of exon 1, 4 and 5 were determined by allele-specific sequencing. A total of 225 unrelated individuals were HLA-B typed by heterozygous sequencing of exons 2 and 3. In the B5/35 CREG, 26 different alleles were identified, whereas 63 non-B5/35 CREG alleles were sequenced. The SBT strategy was proven to be reliable and efficient for high resolution typing of the B5/35 CREG. The nucleotide sequences of exon 1, 4 and 5 were determined for the 26 different B5/35 CREG alleles to establish the level of polymorphism. For seven different alleles, of which the exon 1, 4 and 5 sequences were hitherto unknown, the sequences were elucidated and in agreement with the known B5/35 sequences. Nineteen HLA-B5/35 CREG alleles with previously published exon 1, 4 and 5 sequences were sequenced in at least two individuals. Three new alleles were identified. The first, B*5204, showed a difference at position 200 compared to B*52011, which was previously considered a conserved position. The other two alleles, B*3542 and B*51015, showed exon 2 and 3 sequences identical to B*35011 and B*51011, but differences in exons 1 and 4, respectively. B*3542 had differences at position 25 and 72 and B*51015 showed a difference at position 636. More polymorphism might be present outside exons 2 and 3 than previously thought.     

登革2型病毒E蛋白免疫优势表位的筛选鉴定

目的 用噬菌体展示肽库筛选登革2型病毒(DEN2)E蛋白的抗原表位，并确定该抗原表位性质。方法 以DEN2型特异的E单克隆抗体作为筛选分子，生物淘洗噬菌体随机12肽库，将筛选的噬菌体阳性克隆进行ELISA检测、DNA序列测定及展示肽的氨基酸序列推导，通过噬菌体展示肽序列与DEN2E蛋白的氨基酸一级结构的对比，初步确定E蛋白的抗原表位；用模拟该表位线性序列的合成十肽进行抗体结合试验、噬菌体竞争抑制试验及与DEN感染患者的血清学试验，确定其为免疫优势线性表位。结果 肽库淘洗获得的11个ELISA阳性的噬菌体克隆有相似的结构基序WFKKGSS，其展示肽与DEN2E蛋白390～398 AA序列有3～5个氨基酸相同。对应于DEN2E蛋白390～399AA的合成十肽能与淘洗单抗特异反应，并可抑制噬菌体阳性克隆与该单抗结合。该合成肽与DEN2感染患者血清有较高的免疫反应性。结论 本实验通过噬菌体随机肽库的生物淘洗确定的DEN2E蛋白(E390～398AA)线性序列为免疫优势表位，其对应的合成肽E10可望用于DEN2感染的快速诊断。     

针对单克隆抗体MGb1的重组抗独特型抗体的筛选与鉴定

目的 利用噬菌体呈现技术筛选针对抗胃癌单克隆抗体（简称单抗）MGb1的重组抗独特型抗体（抗-Id），为研制胃癌重组抗-Id瘤苗提供候选分子。方法 以单克隆抗体MGb1免疫Balb／c小鼠，取脾分离mRNA。RT-PCR分别扩增抗体VL和VH cDNA，经linker DNA连接形成ScFv DNA。将scFv DNA与载体pCANTAB5E的连接产物转化于大肠杆菌TG1，经M13KO7感染后，获得重组噬菌体抗体ScFv文库。以单抗MGb1对文库进行4轮淘选后，随机挑取克隆经ELISA筛选呈现抗-Id scFv的噬菌体单克隆，进而经竞争ELISA对其所属抗-Id类型进行初步鉴定。结果 VL和VH cDNA分别约为320和340bp，ScFv DNA约为750bp。抗体ScFv文库经四轮淘选后，在随机筛检的50个克隆中得到18个呈现抗-Id ScFv的噬菌体单克隆。在18个克隆中，有4个呈现β或γ型抗-Id ScFv。结论 经重组噬菌体抗体技术成功地筛选到了针对单抗MGb1的噬菌体呈现型抗-Id ScFv，从而为进一步获得能诱导抗胃癌免疫的抗-Id ScFv奠定了基础。     

利用噬菌体肽库技术获得与人Fas结合的多肽基序

目的　利用噬菌体随机肽库技术获得与人Fas胞外区结合的多肽及其多肽基序 ,观察多肽基序的生物学功能。方法　以人Fas胞外区与IgGFc段的融合蛋白Fas .Fc为筛选配基 ,筛选噬菌体随机九肽库 ;微量淘洗与ELISA相结合鉴定阳性克隆 ,DNA测序和分析。化学合成多肽进行竞争性ELISA以及细胞增殖抑制实验。结果　经过 4轮亲和筛选 ,微量淘洗鉴定 ,获得 4 2个阳性克隆 ;固定ELISA实验显示筛选到的噬菌体短肽能与Fas .Fc特异性结合 ,并呈剂量依赖关系 ;随机选取 13个阳性克隆进行DNA测序 ,其序列及出现几率分别为 :PRKARVDTS(2 / 13)、YKKKSLQVQ (2 / 13)、YKKKSMLQA(2 / 13)、SRKKYDQYA(4/ 13)、YARKIKPTA(2 / 13)和ARKKTEGAG(1/ 13)。经多重序列分析 ,获得多肽基序 : R/KKK A。在ELISA和竞争性ELISA实验中 ,化学合成多肽EGEFYKKKSM LQADPAK (P3)可抑制Fas与抗人Fas单抗Apo 1的结合 ,且呈剂量效应关系 ;P3不能抑制Fas与FasL的结合。细胞增殖实验表明 ,多肽可抑制Jurkat细胞增殖 ,且随多肽剂量的增加而加强。多肽与单抗Apo 1联合作用对Jurkat细胞增殖的影响与单独使用P3没有明显差别。结论　通过噬菌体随机肽库技术获得与Fas结合的多肽及其多肽基序 ,它们可能模拟了抗Fas抗体Apo 1对Fas的结合位点 ,为基于Fas凋     

DLA-DRB1 and DLA-DQB1 histocompatibility typing by PCR-SSCP and sequencing

Abstract: The dog has been an important model for solid organ and hematopoietic stem cell transplantation for over 30 years. Fundamental to the continuing usage of the model is the development of molecular-based histocompatibility typing of donors and recipients. Previous histocompatibility typing methods used in the dog have not been precise enough to identify dog leukocyte antigen (DLA)-matched unrelated dogs. This study was undertaken to begin the process of identifying DLA-matched unrelated dogs. In this study polymerase chain reaction-single-stranded conformational polymorphism is used to separate alleles thereby allowing sequenced-based typing of the two most polymorphic class II genes described to date in the dog - DLA-DRB1 and DLA-DQB1.     

噬菌体随机肽库分析HIV-1 p24抗原表位

目的：利用噬菌体随机肽库分析抗HIV－1核心区抗原p24单抗体在抗原上的识别位点。方法：用抗HIV－1 p24单抗2C7和3H10作为筛选分子，对噬菌体肽库进行生物洗（biopanning)，并通过DN测序、ELISA效价测定等对所获得的噬菌休克隆进行鉴定，最后对合成的7肽位点通过间接ELISA及免疫抑制试验进行血清学分析。结果：序列分析结果表明，单抗2C7和3H10在HIV－1 p24上的抗原识别表位的保守序列分别为DHPXPXX和XXXXKAF。分别合成这2个7肽氨基酸序列P－C1（DHPSPWG）和P－H3（SPWLKAFGGS），并分析其免疫学结合特性，结果表明与P－H3相比，单抗2C7的抗原识别表位P－C1的固相结合特性较好，固相P－C1检测血样，13份抗HIV阳性本中，12份为阳性（检出率为92．3％），19份抗HIV阴性样本中，仅1份为假阳性结果（特异性为94．7％），与P－C1相比，单抗3H10的抗原表位P－H3的固相结合能力极差，但液相结合活性较好，血样与P－H3的抑制试验表明，13份抗HIV阳性样本中12份样本对P－H3的抑制率大于60％（12／13），而9份抗HIV阴性样本中仅1份对P－H3的抑制率大于50％，结论：用抗HIV－1 p24单抗筛选噬菌体随机肽库，获得单抗在p24抗原上的识别表位的氨基酸序列，血清学结果表明这2个抗原表位存在于p24自然抗原上，在抗HIV－1的感染检测中具有潜在的应用价值。     

Functional supertype of HLA-A2 in the presentation of Flu matrix p58-66 to induce CD8+ T-cell response in a Northern Chinese population

The functional supertype of HLA-A2 was investigated in the presentation of the A*0201-restricted Flu matrix p58-66 peptide to activate recall CD8+ T-cell response. In healthy Northern Chinese, the HLA-A2 supertype was mainly composed of the six alleles, A*0201 (26.4%), A*0206 (12.7%), A*0203 (8.2%), A*0207 (7.3%), A*0210 (1.8%) and A*0205 (0.9%), as analyzed by PCR using sequence specific primer (PCR-SSP) and sequence based typing (SBT). The IFN-gamma release Elispot assay was employed to assess effector CD8+ T cells. In A*0201-bearing individuals, the CD8+ T-cell response was potent when stimulated with autologous CD8- PBMCs. The frequency of the effector CD8+ T cells was 96.6% with the magnitude of effector CD8+ T cells of 225 SFC/5 x 104 CD8+ T cells and the RI of 25.7. In non-A*0201 individuals, the effector CD8+ T cells were minimally detectable while the peptide was presented by the autologous CD8- PBMCs. However, the induction of the response of CD8+ T cells obtained from non-A*0201 individuals was remarkably improved when the peptide was presented by autologous dendritic cells instead of CD8- PBMCs. The HLA-A2 alleles possessing cross-reactivity in the peptide presentation were mainly of A*0206 and non-A*0201 heterozygotes of A*0206 and A*0210. Moreover, A*0206 as the HLA-A2 functional supertype was further confirmed by tetramer assay. In two A*0206+ donors with CD8+ T-cell response to the peptide, the CD8+ T-cell frequency assessed by specific binding of peptide HLA-A*0201 tetramer was 4.62% and 1.66%, respectively. Thus, our results have substantiated the immunological relevance of the HLA-A2 supertype, which may benefit the design of peptide vaccines with the potential to be applicable in broader populations.     

Error rate for HLA-B antigen assignment by serology: implications for proficiency testing and utilization of DNA-based typing methods

Until recently, the majority of HLA class I typing has been performed by serology. Expensive commercial typing trays are frequently used for testing non-Caucasian subjects and new strategies using DNA-based methods have been adopted for improving clinical histocompatibility testing results and adapted as supplements in proficiency testing. A double-blind comparison of the typing of HLA-B specificities in 40 samples was carried out between serology and two polymerase chain reaction (PCR) methods, PCR amplification with sequence-specific primers (PCR-SSP) and PCR amplification and subsequent hybridization with sequence-specific oligonucleotide probes (PCR-SSOP). The results demonstrated 22.5% misassignments of HLA-B antigens by serology. There was complete concordance between the results obtained with the two PCR based typing methods. A second panel of 20 donor samples with incomplete or ambiguous serologic results was analyzed by PCR-SSP and SSOP. Both PCR methods identified correctly the HLA-B antigens. Our results suggest that more accurate typing results can be achieved by complementing serologic testing with DNA-based typing techniques. The level of resolution for HLA-B antigen assignment can be obtained by this combination of serology and limited DNA-based typing is equivalent to the HLA-B specificities defined by the WHO-HLA Committee. This level of resolution cannot routinely be achieved in clinical histocompatibility testing or in proficiency testing using serologic reagents only.     

Pulsed-field gel electrophoresis as an epidemiologic tool for enterococci and streptococci

Enterococci (Enterococcus faecium and Enterococcus faecalis) and streptococci such as Streptococcus pyogenes (Group A streptococcus), Streptococcus agalactiae (Group B streptococcus), and Streptococcus pneumoniae are increasing in importance as both hospital-acquired and community pathogens. Emerging resistance and increasing incidence of these organisms has necessitated the analysis of their epidemiologic mechanisms of spread. Pulsed-field gel electrophoresis (PFGE) has emerged as the one of the most widely applicable, reproducible, and stable methods to examine strain identity in bacterial organisms. The procedure used in our laboratory for PFGE typing of whole cell DNA digested with SmaI for enterococci, S. pneumoniae, S. pyogenes, and S. agalacatiae is presented. Issues regarding interpretation are also reviewed and discussed.     

Analysis of HLA-DRB and -DQB gene RFLPs in DR7 homozygous cell lines: associations with Dw11, Dw17 and DB1

The DR7-associated Dw specificities, Dw11, Dw17 and DB1 were investigated with regard to DRB- and DQB-gene polymorphism, as revealed by RFLP analysis using the restriction enzyme TaqI. In the 22 DR7 homozygous cell lines investigated, each of these Dw specificities was found to correlate to one specific RFLP defined DR-DQ haplotype. In addition, a clear linkage disequilibrium to a specific HLA-B locus allele for each Dw specificity was noted, indicating that the Dw subtypes of DR7 often are associated with a conserved HLA-B-DR-DQ haplotype. Only one genetically homozygous cell line, PLH, deviated from these correlations. This cell line, notably derived from an individual with a deletion of the 21-hydroxylase B-gene (21-OHB), caries the HLA haplotype Bw47, DR7, DQw2, DB1, but displayed a DRB RFLP otherwise found in association with Dw17.