中国猪种DQB新等位基因的克隆和分析

克隆和序列分析中国猪种DQB基因cDNA ,为猪异种器官移植的免疫识别机制的研究提供基础。采用RT PCR方法扩增猪DQB基因cDNA ,克隆入测序载体 ,然后进行测序及其序列分析。结果获得具有阅读框架的三个结构和已有序列不同的DQB新等位基因 ,基因序列号分别为AY10 2 4 76、AY10 2 4 77和AY10 2 4 78,其长度为 786个核苷酸 ,除末端终止密码外 ,编码 2 6 1个氨基酸残基。发现了三个猪DQB新等位基因 ,同时发现中国猪种DQB基因及其推导的氨基酸与人相应基因的同源性明显高于小鼠     

中国猪种DQA新等位基因的克隆和分析

目的：克隆和序列分析中国猪种DQA基因cDNA，为猪异种器官移植的免疫识别机理的研究提供基础。方法：采用RT-PCR方法扩增广西巴马猪(GXP)、贵州香猪(GZP)及云南小耳猪(YNP)DQA基因cDNA，克隆入测序载体，然后进行测序及其序列分析。结果：获得具有阅读框架的3个结构和已有序列不同的DQA新等位基因(GXPDQA、GZPDQA和YNPDQA)，基因序列号分别为AY102473，AY102474和AY102475，其长度分别为765、768和768个核苷酸，除末端终止密码外，分别编码254、255和255个氨基酸残基。结论：发现了3个猪DQA新等位基因，同时发现中国猪种DQA基因及其推导的氨基酸与人相应基因的同源性职显高于小鼠。     

湖南大围子猪SLA-DR基因克隆及其生物信息学分析

目的:研究湖南大围子猪SLA-DR基因特性, 评价该猪种在异种器官移植中是否具有应用前景.方法:应用RT-PCR扩增大围子猪SLA-DRA和SLA-DRB基因, 插入pUCM-T载体, 双向测序, 用NCBI中的BLAST和ExPASY软件进行生物信息学分析.结果:大围子猪SLA-DRA和SLA-DRB基因扩增片段大小分别为1 177 bp和909 bp, 均包含完整的开放阅读框, 分别编码252和266个氨基酸残基.生物信息学分析结果表明, 大围子猪SLA-DRA和SLA-DRB与人类相应的DRA、 DRB相比, 氨基酸同源性分别为82%和73%.SLA DR α链与人CD4 分子结合部位介于第124 至136 位氨基酸, 大围子猪SLA-DRA在该结合区域与人类相应的DRA存在两个氨基酸差异, 即:第127位(lle→Val), 第136位(Ser→Thr);SLA DR β链与人CD4 分子结合部位位于第134至148位氨基酸, 大围子猪SLA-DRB在该结合区域与人类相应的DRB相比氨基酸序列完全相同.与国际GenBank 已登录的许多猪种相比, SLA-DRA基因同源性高达100%, 而SLA-DRB基因在各猪种之间具有很高的多态性.结论:克隆湖南大围子猪SLA-DRA和SLA-DRB基因, 该基因与人类相应的HLA-DRA和HLA-DRB基因高度同源, 提示该猪种有望作为异种移植的候选供体.     

中国广西狂犬病毒野毒株（CGX89—1株）糖蛋白基因核…

本报告了中国广西狂犬病毒野毒株（ＣＧＸ８９－１株）糖蛋白基因ｃＤＮＡ的核苷酸序列及其推导的氨基酸序列。ＣＧＸ８９－１株的糖蛋白基因从起始密码ＡＴＧ到终止密码ＴＡＡ共有１５７５个核苷酸残基，可编码形成５２４个氨基酸残基的多肽链，经修饰后形成具有５０５个氨基酸残基构成的狂犬病毒糖蛋白。ＣＧＸ８９－１株的糖蛋白基因和核苷酸组成分别为：Ａ占２７．１１％，Ｔ占２６．２９％，Ｃ占２１．９７％和Ｇ占２４．６３％     

恶性疟原虫GLURP基因的体外扩增，克隆及序列分析

通过体外扩增 ,克隆恶性疟原虫海南 (FCC1 HN)株GLURP基因 ,测定其基因序列 ,了解该基因的结构及在FCC1 HN株与其它分离株间的序列差异。根据GLURP基因已知序列设计合成 3对引物 ,用PCR技术从FCC1 HN株基因组DNA中扩增 3个部分序列重叠的GLURP基因片段 ;并分别克隆入测序用PMD 18T载体。用双脱氧链末端终止法测定GLURP基因序列 ,应用软件辅助分析基因结构及进行同源性比较。恶性疟原虫FCC1 HN株GLURP基因全长 3711bp ,编码 12 36个氨基酸。FCC1 HN株与F32株GLURP基因核苷酸序列同源性为 96 2 7% ;编码氨基酸序列同源性为 95 78%。本文为继续进行FCC1 HN株GLURP抗原的免疫原性和保护性研究奠定基础。     

丙型肝炎病毒E2／NS1区基因cDNA的克隆及序列分析

从北京地区１份丙型肝炎病毒（ＨＣＶ）感染者血清中提取ＲＮＡ，经逆转录和套式聚合酶链反应扩增ＨＣＶＥ２／ＮＳ１区基因约９３０ｂｐ，将其插入至ｐＧＥＭ－Ｔ质粒载体中，利用双脱氧链末端终止法测出该基因５’端４３１ｂｐ的核苷酸序列，将此序列与其它９个ＨＣＶ分离株的相应序列进行比较分析，结果表明，此序列与ＨＣＶⅡ型序列同源性较高，核苷酸水平同源性在８０％以上。由其编码的ＨＣＶ外膜蛋白Ｎ端存在两个高变部位（ＨＶＲ），在ＨＶＲ内、外具有几个保守氨基酸残基和氨基酸区域，它们与外膜蛋白空间构型的维持有关。     

中国广西狂犬病毒野毒株（CGX89－1株）糖蛋白基因核酸序列测定和分析

本文报告了中国广西狂犬病毒野毒株（ＣＧＸ８９－１株）糖蛋白基因ｃＤＮＡ的核苷酸序列及其推导的氨基酸序列。ＣＧＸ８９－１株的糖蛋白基因从起始密码ＡＴＧ到终止密码ＴＡＡ共有１５７５个核苷酸残基，可编码形成５２４个氨基酸残基的多肽链，经修饰后形成具有５０５个氨基酸残基构成的狂犬病毒糖蛋白。ＣＧＸ８９－１株的糖蛋白基因和核苷酸组成分别为：Ａ占２７．１１％，Ｔ占２６．２９％，Ｃ占２１．９７％和Ｇ占２４．６３％。核苷酸序列和巴斯德株（ＰＶ株），国际标准攻击毒株（ＣＶＳ株），中国狂犬病毒疫苗株（３ａＧ株）相比，同源性分别为８４．１％，８３．１％和８４．５％。其推导的氨基酸序列和ＰＶ株、ＣＶＳ株和３ａＧ株相比其同源性分别为９２．４％，８９．７％和８９．５％。ＣＧＸ８９－１株也具有３个Ｎ－糖基化位点，分别位于第３７位、１５７位和３１９位的氨基酸残基上。糖蛋白的膜外区部分重要抗原位点和ＰＶ株、ＣＶＳ株及３ａＧ株具有较高的一致性。     

高原鼠兔血红素氧合酶1的基因克隆及序列分析

 [摘 要] 目的:克隆高原鼠兔血红素氧合酶1（heme oxygenase-1,HO-1）基因cDNA的全长序列,并分析其序列特征,为进一步揭示高原鼠兔低氧适应的分子机制提供有益参考。方法:从高原鼠兔肝组织中提取总RNA,利用逆转录聚合酶链反应（RT-PCR）技术和cDNA末端快速克隆（RACE）技术扩增出HO-1基因cDNA全长序列并进行测序,测序结果采用生物信息学的方法进行分析。结果:克隆所得鼠兔HO-1基因cDNA全长片段大小为1 466 bp,与预期一致,编码区长度为873   bp,编码290个氨基酸（GenBank登录号为：JX035934）;序列分析结果显示,核苷酸序列和推测的氨基酸序列相似性比较,与兔、人、牛、小鼠、大鼠、猪和马的HO-1核苷酸序列的同源性分别为89%、87%、85%、79%、84%、85%和85%,与兔、人、牛、小鼠、大鼠、猪和马的HO-1氨基酸序列的同源性分别为89%、85%、84%、80%、79%、82%和67%,显示出高度保守性。构建的基于氨基酸序列的分子系统进化树聚类结果表明,高原鼠兔与兔的进化距离最近。结论: 本实验首次成功克隆出高原鼠兔HO-1基因cDNA全长序列,为从低氧细胞保护角度,进一步探讨青藏高原土著物种适应高原的分子生物学机制研究提供实验依据。     

福氏2a志贺菌磷酸烯醇丙酮酸盐合成酶基因的克隆与序列分析

目的：福氏2a志贺菌301株染色体编码与代谢相关的酶类基因，分析结构特点并进行同源性比较，以期部分阐明其与大肠杆菌生化特性相似的分子生物学基础。方法：鸟枪法随机克隆福氏2a志贺菌染色体DNA，经探针杂交和末端核苷酸序列测定筛选出带有磷酸烯醇丙酮酸合成酶（pps）完整基因的阳性克隆，利用exouclease Ⅲ制备嵌套缺失体亚克隆，采用双脱氧链末端终止法测定核苷酸序列。结果：福氏2a志贺菌301株pps基因（全长2474bp）与大肠杆菌pps基因核苷酸及氨基酸序列的同源性分别为98．8％和97．6％。编码区上游和下游存在较多变异。福氏2a志贺菌301株pps基因与GenBank中其它菌株pps基因的同源性均低于55％。结论：福氏2a志贺菌pps基因苷酸序列与大肠杆菌pps基因核酸序列高度同源。     

广州地区HCMV低传代临床病毒株UL143基因的多态性研究

目的研究广州地区人巨细胞病毒（HCMV）临床低传代分离株UL143基因的多态性。方法对3株经多重PCR鉴定的HCMV临床低传代分离株进行HCMV UL143基因全序列扩增，扩增产物克隆到pMD18-T载体上测序，并将其序列与GenBank中公布的其它临床分离株UL143基因一起进行分析。结果D3株UL143基因因碱基缺失形成多处终止密码无法产生有功能的蛋白；Toledo株UL143基因开放读码框由279个核苷酸组成．编码蛋白由92个氨基酸残基组成：其它临床分离株UL143基因开放读码框均由252个核苷酸组成。DNA序列比较保守，变异均为碱基替换。编码蛋白由83个氨基酸残基组成，氨基酸序列也很保守，不同临床分离株氨基酸变异率为1．2％-2．4％：HCMV UL143蛋白翻译后修饰位点在除Toledo株之外的所有分离株中均高度保守．没有缺失或新增；不同临床分离株UL143蛋白二级结构有所不同；除Toledo株外，其余分离株UL143蛋白的等电点均为8．75。结论临床低传代分离株HCMV UL143基因DNA及其编码产物的氨基酸序列极为保守。但仍存在一定多态性。     

近交系海南五指山猪SLA经典I类和II类分子序列分析

为筛选适合我国异种移植研究的猪种提供参考依据 ,采集近交系海南五指山猪种全血标本 6例 ,利用RT PCR扩增得到cDNA产物 ,纯化、克隆、测序得到SLA经典I类P1、P14座位和II类DQA、DQB、DRA、DRB座位基因序列共 6个。与Genbank中相关序列进行比较分析发现 :所得序列是SLAI类和II类基因的新等位基因。在中国非协调性猪 人异种移植中 ,海南五指山猪种在与人类MHC遗传基因水平相似性方面有一定优势 ,可作为备选猪种对相关基因进行必要的修饰和改造     

Novel SLA-DR alleles of three Chinese pig strains and the related function in human T cell response

To elucidate the structures of SLA-DR (swine leukocyte antigen DR) genes of three Chinese pig strains (Gz, Bm and Yn), the SLA-DRA and SLA-DRB cDNA were amplified by RT-PCR and subjected to determine the sequences. The whole structures of SLA-DRA alleles are identical among three strains, consisting of 759 nucleotides including an open reading frame (ORF), and are shared with those reported from NIH minipigs SLA-DRAc and SLA-DRA^d. The same length of the ORF-containing SLA-DRB genes of three Chinese pig strains was also identified. They are composed of 801 nucleotides encoding a xenogeneic antigen molecule of 266 amino acid residues. The nucleotide sequences of the SLA-DRB genes, however, are different when compared either among the three strains or with the published data of SLA-DRB sequences, which allowed our novel SLA-DRB alleles receiving their accession numbers AY102479, AY102480 and AY102481 from the GenBank. This study further reveals that the phylogenic homologies of MHC DR or DR-like genes in structures of nucleotides and deduced amino acids between Chinese pigs (SLA) and human (HLA-DRBI*0901) are better than those between pigs and mice (H-2^b Eβ). High similarities were also found for DRα-DRβ heterodimers between Chinese pigs and human in terms of amino acids sequences critical for binding with human CD4 coreceptor molecule, which are better than those between SLA-DR and H-2 I-E molecules. A functional test indicated that, by cotransfection with Bm-DRA and Bm-DRB genes, the Bm-DR molecule-expressed L929 cells could stimulate human T cells quite well in a xenogeneic reaction in presence of human APCs.     

The major histocompatibility complex in swine

Summary: In swine, the major histocompatibility complex ( Mhc ) or swine leukocyte antigen ( SLA ) is located on chromosome 7 and divided by the centromere. Thus, the telomeric class I and more centromeric class III regions are located on the p arm and the class II region is located on the q arm. The SLA region spans about 2 Mb, in which more than 70 genes have so far been characterized. Despite its division by the centromere, the spatial relationships between the genes in the class II and class III regions, and between the well-conserved non-class I genes of the class I region, are similar to those found in the human HLA complex. On the other hand, no orthologous relationships have been found between the Mhc class I genes in man and swine. In swine, the 12 SLA class I sequences constitute two distinct clusters. One chister comprises six classical class 1-related sequences, while the other comprises five class I-distantly related sequences including two swine homologous genes of the HLA Mhc class I chain-related gene ( MIC ) sequence family. The number of functional SLA classical class I genes, as defined by serology, probably varies from one to four, depending on the haplotype. Some of the SLA class I-distantly related sequences are clearly transcribed. As regards the SLA class II genes, some of them clearly code for at least one functional SLA-DR and one SLA-DQ heterodimer product, but none code for any DP product. The amino acid alignment of the variable domains of 33 SLA classical class I chains, and 62 DRβ and 20 DQβ chains confirmed the exceptionally polymorphic pattern of these polypeptides. Among the class II genes, the genes are either monomorphic, like the DRA gene, or oligomorphic, like the DQA genes. In contrast, the DRB and DQB genes display considerable polymorphism, which seems more marked in DRB than DQB genes.     

Sequence-based characterization of the eight SLA loci in Korean native pigs

Eight swine leucocyte antigen (SLA) gene (SLA-1, SLA-2, SLA-3, SLA-6, DRA, DRB1, DQA, DQB1) alleles were identified using sequence-based typing method in three Korean native pigs used for breeding at the National Institute of Animal Science in Korea. Six new alleles in class I genes and three new alleles in class II genes have been identified in this breed and can give valuable information for xenotransplantation and disease resistance.     

Complete cDNA sequences of the HLA-DRB1*14011, *1402, *1403 and *1404 alleles

Sequencing studies of HLA class II molecules have been focused almost exclusively on the highly polymorphic exon 2. In this study the complete cDNA sequence of four alleles of the DR14 lineage (DR52 group) are reported for the first time. The HLA-DRB1*1402 and *1403 sequences were shown to be identical to the previously determined DRB1*13011 sequence, also of the DR52 group, in exons 1, 3, 4, 5 and 6. HLA-DRB1*14011 and *1404 were identical to DRB1*13011 in exons 1, 4, 5 and 6 sequences while they showed specific features within their exon 3 sequence. Both alleles showed a synonymous substitution at the third base of codon 114. However, DRB1*14011 also has a non-synonymous substitution at the first base of codon 112 which results in a histidine to tyrosine substitution. This is a novel substitution as Histidine 112 is conserved in all known HLA class II B genes.     

Sequence‐based characterization of swine leucocyte antigen alleles in commercially available porcine cell lines

A total of 53 alleles at five highly polymorphic swine leucocyte antigen (SLA) loci (SLA‐1, SLA‐3, SLA‐2, SLA‐DRB1, and SLA‐DQB1) were identified in eight commercially available porcine cell lines (ESK‐4, LLC‐PK1, MPK, PK13, PK15, PT‐K75, SK‐RST, and ST). This information is essential for the use of these cell lines to understand the role of SLA genes and proteins in swine models of transplantation, xenotransplantation, and in swine immune responses to infectious diseases and vaccines. The ready availability of these cell lines also makes them a good source of reference DNA for SLA allele typing.     

Motif analysis of HLA class II molecules that determine the HPV associated risk of cervical carcinogenesis

In previous studies, the HLA class II haplotype HLA DRB1*0401-DQB1*0301 was shown to correlate with susceptibility to HPV infection, CIN and cervical cancer while DRB1*0101-DQB1*0501 indicated protection. The present study was designed to identify naturally processed peptide sequences bound to the susceptibility and protective HLA DR-DQ molecules, and use this for T-helper epitope prediction from HPV 16. The HLA class II molecules were obtained by immuno-affinity purification of Epstein-Barr virus B lymphoblastoid cell lines (BCL) homozygous for HLA DQA1*0301-DQB1*0301 and HLA DQA1*0101-DQB1*0501. Peptide pools eluted from the HLA molecules were sequenced by Edman degradation. On the basis of the peptide sequence data obtained, the E6, E7, L1 and L2 proteins of HPV 16 were examined to identify sequences which are likely to bind to HLA DQB1*0301 and DQB1*0501. In addition, motif prediction as well as the binding affinity of predicted peptide motifs for HLA DRB1*0401 and DRB1*0101, the DR alleles associated with susceptibility and protection respectively, was accomplished using published data and a prediction algorithm for the naturally processed peptide sequences bound to these molecules. The HLA DQB1*0501 peptide ligand sequence showed that proline gives an outstanding signal at position 2, Asn/Arg at P1, aliphatic/aromatic amino acids in the central portion, a hydrophobic cluster at P5 with a small contribution by small polar residues and another cluster of aromatic residues towards the C-terminus. The HLA DQB1*0301 sequence also showed that proline gives an outstanding signal at position 2, Thr/Arg at P1, aliphatic/aromatic amino acids in the central portion and an aliphatic cluster with a small contribution by small polar residues at P5. There were no differences in the number of HPV peptides that were predicted as being capable of binding to HLA DQB1*0301 and HLA DQB1*0501, but more HPV peptide motifs were predicted to bind with high affinity to HLA DRB1*0101 than DRB1*0401. The results suggest that HPV 16 peptide epitopes bind with higher affinity to the protective than to susceptible HLA DR-DQ molecules which may lead to a more effective immune response.     

Humanized transgenic mice expressing HLA DR4-DQ3 haplotype: reconstitution of phenotype and HLA-restricted T-cell responses

Many autoimmune conditions have close genetic linkages to particular human histocompatibility leukocyte antigen (HLA) class II genes. With the aim of establishing a murine model of autoimmune disease, we have generated an HLA DR4-DQ3 haplotype transgenic (Tg) mouse that expresses a 440-kb yeast artificial chromosome harbouring DRA, DRB1*040101, DRB4*010301, DQA1*030101, DQB1*0302 and all the internal regulatory segments. This Tg mouse line was crossed to human CD4 (hCD4) Tg mice and endogenous class II knockout mice (I-A(o/o) and I-E(o/o)) lines to generate a DR4-DQ3.hCD4.IAE(o/o) Tg line. The Tg DR and DQ molecules are expressed on the physiological cell types in these animals, i.e. on most B cells (>85%), dendritic cells (DCs) and macrophages but not on T cells, with levels of expression comparable with those of human B cells (where DR > DQ expression). The DR4/DQ3 transgenes fully reconstituted the CD4 T-cell compartment, in both the thymus and the periphery, and the analysis of the T-cell receptor repertoire in the Tg mice confirmed that these class II molecules were able to mediate thymic selection of a broad range of Vbeta families. HLA DR4- and DQ3-restricted T-cell responses were elicited following immunization with known T-cell determinants presented by these molecules. Furthermore, the DR4-DQ3-restricted CD4(+) T cells conferred protective antibody-mediated immunity against an otherwise lethal infection with Salmonella enterica var. typhimurium. These new DR4-DQ3 Tg mice should prove to be valuable tools for dissecting the importance of this class II haplotype in autoimmune disorders like rheumatoid arthritis.     

HLA-DR51 expression failure caused by a two-base deletion at exon 2 of a DRB5 null allele (DRB5*0110N) in a Spanish gypsy family

Here we describe a new HLA class II null allele at the DRB5 gene. Serologic HLA typing of a Spanish gypsy family rendered the following paternal haplotype: A2-Cblk-B52-Bw4-DR15-DQ5. However, DNA typing demonstrated the presence of a DRB5 gene in the haplotype DRB1*1502-DRB5*0102-DQB1*05031. Complete DRB5 cDNA sequencing revealed a DRB5*0102 allele with a deletion of two nucleotides at exon 2 (239-240) in codon 80. This change generates a frame shift leading to a stop codon at position 86, and could explain the lack of DR51 protein at the cell surface. This is the second DRB5 null allele described together with DRB5*0108N, raising the number of HLA alleles with an expression disorder.     

Sequence variation in Japanese HLA-DRw8 specificity.

There are four DRw8 haplotypes with different DQ alleles in Japanese: DRw8-DQw6 (w1), DRw8-DQw4, DRw8-DQw8(w3), and DRw8-DQw7 (w3). We previously reported the nucleotide sequence of DRB1 gene of DRw8-DQw6(w1) and it was named DRB1*08032. The nucleotide sequences of the other DRw8 DRB1 alleles and their correspondence to internationally recognized DRw8 subspecificities were still unclear. We have cloned these DRB1 genes and determined the nucleotide sequences. The comparison of the sequences with the published sequences revealed that the differences were occurred at two amino acid positions, and these four haplotypes are classified in two groups: (a) DRw8-DQw6(w1) and DRw8-DQw7(w3), and (b) DRw8-DQw4 and DRw8-DQw8(w3). The DRB1 molecules of DRw8-DQw6(w1) and DRw8-DQw7(w3) have Ser57 and Ile67, and those of DRw8-DQw4 and DRw8-DQw8(w3) have Asp57 and Phe67. The former has the same sequence as that of DRB1*08032, and the latter is same as that of DRB1*0802. The classification corresponds to the serologic subtyping, which divides DRw8 into DR8.1 and DR8.2, reported in the 10th Japan HLA Workshop.