采用PCR-RFLP技术检测中国人HLA纯合细胞DQ基因多态性

本文采用PCR-RFLP方法对7株中国人HLA纯合细胞的DQA1、DQB1基因作基因分型,检出了这些细胞株的DQ基因类型,证实了中国人群存在着一种DRw12与DQA1*0601-DQB1*0301连锁的新单倍型。     

用细胞内细胞因子染色方法检测HLA-A*02个体CD8+T细胞的免疫应答

目的 :用细胞内细胞因子染色方法 (ICCS)检测不同HLA A 0 2个体的CD8+ T细胞对流感病毒多肽的免疫应答状况。方法 :采用SSP法确定HLA A 0 2亚型 ;分离HLA A 0 2个体的外周血单个核细胞 (PBMC) ,与流感病毒特异的HLA A 0 2 0 1限制性CTL表位多肽 (IMP5 8 6 6 ,GILGFVFTL)孵育 ,ICCS检测CD8+ T细胞分泌细胞因子IFN γ、IL 2和TNF α。结果 :发现HLA A 0 2不同亚型的人PBMC对流感病毒多肽均具有记忆性免疫应答。结论 :①ICCS方法可特异性定量检测T细胞活化状态 ;②HLA A 0 2人群中普遍存在对IMP5 8 6 6多肽的记忆性免疫应答 ;③对于HLA A 0 2 0 1限制性多肽 ,与其它不同HLA A 0 2亚型间存在交叉反应性。     

用PCR-RFLP方法探测MEG白血病细胞HLA-Ⅱ基因型

研究MEG白血病细胞系表达HLA Ⅱ类抗原类型 ,提高对该细胞的HLA Ⅱ基因型的认识。旨在从分子遗传学水平上研究HLA Ⅱ类抗原与急性巨核细胞白血病发病机理的关系。采用聚合酶链反应 限制性片段长度多态性 (PCR RFLP)技术分析MEG白血病细胞系HLA Ⅱ类抗原类型。结果是MEG白血病细胞系的HLA Ⅱ类抗原的等位基因型分别是 :DRB1 14 0 6 0 4 10 ,DQB1 0 4 10 0 4 0 1,DPB1 0 5 0 1 0 5 0 1。该项研究提高了对巨核细胞白血病细胞系HLA Ⅱ基因型的认识 ,并为临床上进一步探讨HLA Ⅱ基因型与急性巨核细胞白血病致病基因之间…     

应用聚合酶链反应-限制性片段长度多态技术快速检测中国耳聋人群基因突变热点

目的 通过筛查耳聋基因热点突变:GJB2基因的235delC、SLC26A4基因的IVS7-2A>G和线粒体12S rRNA(12S)基因1555 A>G达到快速诊断耳聋患者.方法 多重PCR扩增包括GJB2、SLC26A4及12S基因的3个片段,限制性片段长度多态分析是否存在相应位点的突变.结果 200例耳聋患者中,共检测出235delC纯合突变18例,杂合突变18例;IVS7-2A>G纯合突变2例,杂合突变13例;1555 A>G突变8例.检测结果均与测序结果相符合.3个热点突变基因的致病单体的检出率为21.7%,基因诊断率为14%.结论 应用聚合酶链反应-限制性片段长度多态技术检测耳聋患者的热点突变是一种快速、简便、高效、经济的耳聋致病基因筛查的方法.     

中国汉族人HLA纯合细胞及绒猴细胞系DP基因结构分析

应用聚合酶链反应、基因克隆和末端终止技术，测定了分属ＤＲ９、ＤＲ１１和ＤＲ１２的６个中国汉族人ＨＬＡ纯合细胞ＤＰＢ１基因第二外显子的核苷酸序列。结果表明，６个纯合细胞分别带有４种等位特异性ＤＰＢ１＾＊０２０１２、ＤＰＢ１＾＊０２０２、ＤＰＢ１＾＊０５０１、ＤＰＢ１＾＊１３０１，基因结构和白种人相同，因而采用国外参考试剂和探针对中国人标本作ＤＰＢ１基因分型是可行的。同时，首次揭示了绒猴ＤＰＢ１基因第     

中国汉族人HLA纯合细胞及狨猴细胞系DP基因结构分析

应用聚合酶链反应、基因克隆和末端终止技术，测定了分属ＤＲ９、ＤＲ１１和ＤＲ１２的６个中国汉族人ＨＬＡ纯合细胞ＤＰＢ１基因第二外显子的核苷酸序列。结果表明，６个纯合细胞分别带有４种等位特异性ＤＰＢ１＊０２０１２、ＤＰＢ１＊０２０２、ＤＰＢ１＊０５０１、ＤＰＢ１＊１３０１，基因结构和白种人相同，因而采用国外参考试剂和探针对中国人标本作ＤＰＢ１基因分型是可行的。同时，首次揭示了狨猴ＤＰＢ１基因第二外显子序列，该序列和人类ＤＰ基因同源性为６９．９％。     

血清学指定HLA-B座位纯合型误差分析及HLA-B座位表达变异检测的意义

目的 :用DNA分型法对HLA B座位抗原纯合型进行分型 ,探明是否存在HLA B基因发生变异引起结果差异。方法 :经血清学鉴定HLA B座位纯合型样本 75例 ,用PCR SSP法进行DNA分型和表达变异筛查。结果 :75例血清学鉴定为纯合型后经DNA分型证实有 12例存在第二基因 ,经PCR SSP法进行变异筛查发现有 1例存在第四外显子额外胞嘧啶插入 ,经DNA分型发现的第二基因实为沉默基因。结论 :用PCR SSP法进行HLA B基因分型可弥补血清学方法的不足 ,同时筛查表达变异使分型结果更加准确 ,能有效地指导临床应用。     

中国人群Auberger抗原基因多态性研究

目的 对中国人群Latheran血型系统中Auberger抗原的摹因多态性分布进行研究,建立起稳定、准确的Auberger抗原分子生物学的检测方法.方法 随机采集162名非血缘关系无偿志愿捐血者外周血样,直接进行Auberger血型抗原基因的第12外显子测序分析.对新位点突变应用聚合酶链反应-限制性片段长度多态性法电泳分析.结果 在162人份标本中Aua+b-(nt 1615A)119人,Aua+b(nt 1615A/G)40人,Aua-b+(nt 1615c)3人,Aua和Aub的基因频率分别为0.8580和0.1420.在1例基因为Aua纯合型个体中,出现nt1595G＞T突变,PCR产物经Hha Ⅰ限制性内切酶内切后,突变型和野生型的个体分别被分为不同片段,可证实该突变存在.结论 建立起Auberger抗原分子生物学的检测方法,得到中国人群Auberger抗原基因的多态性分布,发现一个新的Lutheran等位基因(GenBank注册号:EU2N1043).     

中国南方汉族人群HLA-Cw座位基因多态性分析

目的 分析中国南方汉族人群HLA－Cw座位基因多态性。方法 采用ARMS／PCR技术对60名随机选择的南方汉族健康个体作HLA－Cw基因分型。结果 共检出12种HLA－Cw等位基因或等位基因组（allele group)，其中HLA－Cw*07、＊01、＊03为该人群最常见HLA－Cw基因，频率之和为0．6932；证实该群体中HLA－Cw＊08频率为0．1056，检出率明显高于血清学分型；检出HLA－Cw*1202、＊1203、＊14、＊15等4种经典血清学分型不能鉴定的HLA－Cw基因，频率之和为0．0673。结论 提供了中国南方汉族人群HLA－Cw座位基因频率正常值，证实ARMS／PCR作HLA－Cw分型具有准确、快捷的优点。     

中国人D13S31等位点的多态性及其在Wilson‘s病家系中的应用

本文对９０名无亲缘关系的正常中国人进行了Ｄ１３Ｓ３１和Ｄ１３Ｓ２５区域的限制性片段长度多太性分析（ＲＦＬＰ），表明中国上述位点的多态等位片段与西方人相同，但两者各等位片段的频率差异明显，杂合率亦不同。应用上述位点对４个Ｗｉｌｓｏｎ’ｓ病（ＷＤ）家系进行多位点连锁分析，证实它们可用于ＷＤ的症状前诊断。     

HLA-DR generic typing by AFLP.

We have described a practical and inexpensive method whereby any individual can be typed and assigned to any of the 14 generic HLA-DR types: DR1, DRw15, DRw16, DRw17, DRw18, DR4, DRw11, DRw12, DRw13, DRw14, DR7, DRw8, DR9 and DRw10. Previous methods to type these specificities include - among others - serology, conventional RFLP, PCR-oligonucleotide typing, and a PCR-RFLP method useful for typing homozygous individuals. In the method reported here, DNA is amplified with a set of group-specific primers and then restricted with a number of endonucleases. Six group-specific pairs of primers have been chosen to avoid cross-amplification with other DRB alleles, including DRB3, DRB4 and DRB5 alleles, and to anneal at uniform temperature: 61 degrees C. Endonucleases were chosen to generate unique patterns of easily recognizable bands that led to unequivocal assignments of HLA-DR generic types in heterozygous as well as homozygous individuals. This technique involves two steps: 1) Amplification of DNA with six different pairs of primers where DR1, DR4 and DRw10 types can be assigned at once, and 2) Endonuclease digests of amplified DNA to assign DR7, DRw8, DR9, DRw11, DRw12, DRw13, DRw14, DRw15, DRw16, DRw17 and DRw18 types. Individuals carrying any combination of all alleles published so far can be typed by this method. The ease, low cost and reliability of this method are discussed.     

Direct evidence for a functional role of HLA-DRB1 and -DRB3 gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T lymphocytes

The contribution of the HLA-DRB1, -B3, and -B5 gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T cells isolated from an atopic individual (HLA-DRw12, DR7; DRw52b) with perennial rhinitis was investigated. Using a panel of histocompatible and histoincompatible accessory cells, the restriction specificity obtained for a long term T cell suggested that a component of the dust mite reactive repertoire recognized antigen in association with DRB3 gene products. Oligonucleotide DNA typing of the presenting cell panel demonstrated a correlation between the DRw52b allele and T cell responsiveness. Murine fibroblasts expressing DRw52b, but not DRw52a or -c molecules, presented antigen to both the T cell line and cloned T cells (DE26) derived from the line, indicating that the supertypic specificity DRw52b was able to restrict recognition of dust mite antigens. Additional T cell clones (DE9 and DE41) also isolated from the line were restricted by the products of the B1 gene locus (DRw12B1) as determined by murine fibroblasts transfected with the appropriate HLA-DR genes. Clone DE9 was degenerate in its restriction specificity, also recognizing dust mite presented by accessory cells expressing the DR2 subtypes. Presentation by fibroblasts transfected with DRw12B1, DR2Dw2B5 genes and EBV-transformed B cell lines expressing DR2DW21B1 and -B5 indicated that the functional site restricting recognition may be associated with residues 70 and 71 of the DR beta chain helical wall of the antigen combining site. Furthermore, we have recently demonstrated that both T cell clones DE9 and DE26 induce allergen dependent IgE synthesis in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new supertypic HLA class II determinant (PL2) differentially expressed with DR7, DRw11(5), DRw13(w6), DRw14(w6), DR3, and DR2 and biochemically localized to DR7 molecules

A monoclonal antibody, PL2, has been produced that reacts with a new supertypic determinant expressed on the peripheral blood B lymphocytes and B-leukemic cells (B-CLL) from all individuals who are HLA-DR7 and some individuals who are HLA-DR5 positive. The genetic linkage of the PL2 determinant to the HLA region was demonstrated by family segregation studies. When cultured Epstein-Barr virus (EBV) transformed B cell lines were examined, PL2 was again found to be expressed on all cell lines homozygous for HLA-DR7 and the DRw11(5) subtype of HLA-DR5 positive cells, while one DRw12(5) cell line was negative, suggesting PL2 may distinguish between these DR5 subtypes. In addition, using the panel of EBV-transformed B-cell lines, PL2 was also found to be weakly expressed on HLA-DRw14(w6), -DRw13(w6), -DR3, and -DR2 positive cells but was completely absent from HLA-DR1 and -DR4 positive cells, and is probably absent also from DRw8- and DRw10-positive cells. From titration analysis and quantitative absorption studies the PL2 determinant was found to be expressed at quantitatively different levels in the following order: DR7 greater than DRw11, DRw14 greater than DRw13 greater than DR3 greater than DR2. The molecules carrying the PL2 determinant on DR7 cells have been characterized biochemically to be a subpopulation of HLA class II molecules recognized by the DR specific monoclonal antibody, L243. Furthermore, by two-dimensional gel analysis, PL2 immunoprecipitated only two of three beta chains associated with the DR-apha chain, which are the same two chains that carry the DR7 allodeterminants.(ABSTRACT TRUNCATED AT 250 WORDS)     

HLA-DR generic typing by AFLP

Abstract: We have described a practical and inexpensive method whereby any individual can be typed and assigned to any of the 14 generic HLA-DR types: DR1, DRwl5, DRwl6, DRwl7, DRwl8, DR4, DRwll, DRwl2, DRwl3, DRwl4, DR7, DRw8, DR9 and DRw10. Previous methods to type these specificities include - among others - serology, conventional RFLP, PCR-oligonucleotide typing, and a PCR-RFLP method useful for typing homozygous individuals. In the method reported here, DNA is amplified with a set of group-specific primers and then restricted with a number of endonucleases. Six group-specific pairs of primers have been chosen to avoid cross-amplification with other DRB alleles, including DRB3, DRB4 and DRB5 alleles, and to anneal at uniform temperature: 61deg;C. Endonucleases were chosen to generate unique patterns of easily recognizable bands that led to unequivocal assignments of HLA-DR generic types in heterozygous as well as homozygous individuals. This technique involves two steps: 1) Amplification of DNA with six different pairs of primers where DR1, DR4 and DRw10 types can be assigned at once, and 2) Endonuclease digests of amplified DNA to assign DR7, DRw8, DR9, DRw11, DRw12, DRw13, DRw14, DRw15, DRw16, DRw17 and DRw18 types. Individuals carrying any combination of all alleles published so far can be typed by this method. The ease, low cost and reliability of this method are discussed.     

A cytotoxic monoclonal antibody (HU-39) that detects DRw8 + DRw12

A monoclonal antibody, HU-39, was produced by immunizing BALB/c mice with a cultured human B lymphoblastoid cell line, Shi-C3 (HLA-A24, A31, B51, Bw52, DR2, DRw12, DQw1, DQw3). Utilizing the complement-dependent cytotoxicity test, HU-39 was found to detect a polymorphic determinant common to HLA-DRw8 and HLA-DRw12, a split antigen of HLA-DR5. Although HU-39 reacted with the cells from all of nine DRw12 positive individuals, the cells from only 18 out of 21 DRw8 positive individuals reacted with HU-39 and the remaining three were negative for HU-39. The cytotoxicity of the antibody was reduced after the surface HLA-DR molecules of two cell lines, GI and EBV-Sh, typed as DRw8 and DRw12, respectively, were masked with F(ab')2, of anti-HLA-DR monoclonal antibody. The results of the sequential coprecipitation test and the two-dimensional gel electrophoresis by using EBV-Sh also indicated that HU-39 preferentially recognizes an epitope borne on the DR molecules, but not on the DQ molecules. Thus, HU-39 appeared to be of great value as a tissue typing reagent to define DRw8 and DRw12, the latter of which had been difficult to assign because of the lack of monospecific alloantisera.     

HLA-DRB1 genotyping by modified PCR-RFLP method combined with group-specific primers

We previously introduced HLA-DQA1, -DPB1 and DQB1 genotyping with the modified PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or alternatively no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. In this study, 43 HLA-DRB1 alleles, excluding DRB1*1103 and *1104 for which no restriction enzymes are available to distinguish each from the other, could be defined by this modified PCR-RFLP method combined with 7 pairs of group-specific primers. It is impossible to distinguish DRB1*0701 and DRB1*0702 as they are identical for the second exon of DRB1. For DR1-DRB1, DR2-DRB1, DR4-DRB1, DR7-DR1, DR9-DRB1, DRw10-DRB1 or DRw52 associated antigens (DR3, w11, w12, w13, w14, and DRw8)-DRB1 gene amplification, the second exon of the DRB1 gene was selectively amplified using each group-specific primer from genomic DNAs of 70 HLA-homozygous B-cell lines and healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA, although some alleles can be distinguished only after examination of RFLP band patterns generated and in some cases using double digestion technique with two restriction enzymes. This modified PCR-RFLP method can be successfully applied to all possible DRB1 heterozygotes, despite the fact that 15 pairs of heterozygotes among them cannot be distinguished theoretically by the PCR-SSO method, because the PCR-RFLP method can tell whether two polymorphic sites are linked to each other (cis position) or located on a different chromosome (trans position) by checking the length of RFLP bands generated with double digestion. Thus, the PCR-RFLP method is technically simple, practical and inexpensive for determination of the HLA-DRB1 alleles for routine HLA typing work.     

RFLP analysis of HLA-DR5 haplotypes

The analysis of HLA-DR5 haplotypes unravelled a new DRB3 polymorphism and permitted the identification of various associations between alleles of the DRB1 and DRB3 loci. This new polymorphism consists of a 10.5 kb Taq1 restriction fragment which was encountered in an African-American family (JS). In Caucasoids, the DRw11 allele has been previously observed only in association with the DRw52b allele. RFLP and oligonucleotide typing of HLA-DRw52 alleles associated with DRw11 showed, however, that 4 Caucasoid individuals from our panel carried the DRw52a allele and 1 the DRw52c allele. Similarly, DRw12, which is usually associated with DRw52b, was encountered with DRw52a in 1 Chinese and with DRw52c in an African-American and a Chinese panel member. The study of DRB3 alleles associated with DRw11 and DRw12 indicates that, similar to serology, RFLP studies become particularly informative when individuals of different races and ethnic origins are studied.     

Structural polymorphism of the beta chain of human HLA-DR antigens

[³H]Phenylalanine-labeled HLA-DR antigens were isolated from four human cultured B lymphoid cell lines RPMI 1788 (DRw4,7), RPMI 4098 (DRw2,6), RPMI 6410 (DRw4,6) and Victor (DRw4,6) by indirect immunoprecipitation with xenoantiserum. The and β chains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following digestion with trypsin the peptides were separated by high pressure liquid chromatography. The four HLA-DR chain peptide maps were nearly identical. The HLA-DR chains isolated from the phenotypically distinct cell lines, RPMI 1788 (DRw4,7) and RPMI 4098 (DRw2,6), had strikingly different tryptic peptide profiles whereas the tryptic peptides maps of HLA-DR β chains isolated from the phenotypically identical cell line, RPMI 6410 (DRw4,6) and Victor (DRw4,6), were homologous in 14 out of 19 total peptides, suggesting that the molecular basis for the serologically defined polymorphism of HLA-DR antigens resides in the β chain. The cell line RPMI 4098 was also labeled with [³H]glucosamine and the and β chains of HLA-DR antigen were isolated. Only one tryptic glucosamine-containing peptide was detected in the chain tryptic digest while two peptides were detected in the tryptic digest of the β chain, suggesting that the chain has one carbohydrate moiety and the β chain two carbohydrate moieties.     

Quantitative and qualitative differences in HLA-DR molecules correlated with antigen-presentation capacity

The monoclonal antibodies 7.3.19.1 (anti-DRw52-like) and B8.11.2 (anti-DR framework) were used for the isolation and characterization of HLA class II molecules expressed by HLA-DR3 and DR5 homozygous B cell lines. Sequential immunoprecipitation studies demonstrated that from these cells class II molecules can be isolated which are characterized by the presence or absence of DR framework (DR) and DRw52-like (DRw62) determinants: (DR+, DRw52+), (DR+, DRw52-) and (DR-, DRw52+). The DR3 donor cells appeared to express only the (DR+, DRw52+) and (DR-, DRw52+) class II molecules whereas DR5-positive cells express only the (DR+, DRw52+) and (DR+, DRw52-) class II molecules. Besides qualitative differences some of the above-mentioned molecules appeared to differ in their levels of expression. To investigate whether this might have functional implications, cells with the HLA-DR3 and -5 haplotypes were used to present antigen purified protein derivative of tuberculin (PPD) to PPD-specific T cell lines and the blocking capacity of the two monoclonal antibodies 7.3.19.1 and B8.11.2 was determined. A remarkable correlation was observed between the type of class II molecule blocked by these monoclonal antibodies and its quantitative expression. However, (DR-, DRw52+) molecules, clearly expressed by DR3 cells, were not involved in the presentation of PPD. This indicates that not only quantitative but also qualitative aspects may play a role in the selection of the type of class II molecule that will be involved in antigen presentation.