HLA-Ⅱ类基因DRα及DRB1*0401转基因小鼠的制备

利用受精卵原核的显微注射技术,将人类HLA-Ⅱ类DRα及DRB1 *0401两种基因,显微注射至C57BL/6×DBA/1杂交小鼠受精卵中;并移植至假孕受体鼠的输卵管内.实验先后有8只鼠妊娠,稳定遗传五代,经PCR检测95只HLA-DRα、DRB1*0401阳性.α-32P-dCTP斑点杂交与Southem印迹杂交鉴定出68只整合含有DRα及DRB1 *0401混合型的转基因小鼠,有效率为20.9%.经Northern杂交…和RT-PCR检测,其HLA-DRα、DRB1 *0401基因在脾脏和肾脏中均有表达.结果说明,携带人类HLA-Ⅱ类DRα和DRB1* 0401基因的转基因动物模型构建成功.     

β-葡萄糖苷酶Bgl2238生物信息学分析

利用生物信息学方法对β-葡萄糖苷酶Bgl2238的氨基酸序列进行分析。采用Prot Param、DNAMAN、Multicoil、SOPMA、PROSAN等软件对其理化性质、亲/疏水性、不稳定指数、PEST序列、蛋白质二级结构及蛋白质修饰位点等重要参数进行预测,并对其三级结构进行同源建模及模建结果质量的评估。结果发现:Bgl2238由745个氨基酸组成,蛋白质序列中α-螺旋占36.64%,β-延伸链占19.06%,β-转角占9.40%,无规则卷曲占34.90%,存在14段非PEST序列,具有41个不同蛋白质修饰位点。采用在线网站SWISS-MODEL建模得到了Bgl2238的三级结构,分析蛋白质可及性并分析得到Ramachandran图,检测结果说明三维结构是合理的。本研究结果为β-葡萄糖苷酶Bgl2238进一步研究水解功能及提高其酶活性奠定了理论基础。     

源于红树林的新型脂肪酶Lip906生物信息学分析

本研究利用生物信息学在线软件对脂肪酶Lip906的二级结构和模体信息进行预测,同时对其三级结构进行同源建模和模建结果质量评价,预测该蛋白质的活性位点信息,旨在从蛋白质序列特征和分子结构水平理解其在酯类水解过程中的作用。结果表明,模建的Lip906蛋白结构品质较高,具有7段α-螺旋和2组β-折叠结构,是一个典型的α/β类蛋白,表面呈弱负电势分布;Lip906蛋白具有5个不同模体,可能参与不同生化反应或执行不同的功能。这些研究结果对理解Lip906蛋白功能以及配基结合位点定位非常重要,也为脂肪酶Lip906的突变设计提供了理论基础。     

超抗原葡萄球菌肠毒素A、B和C1的T细胞抗原HLA表位预测

为预测超抗原葡萄球菌肠毒素（SE）家族中的SEA、SEB和SEC1的HLAⅠ和HLAⅡ抗原结合表位,并对其活化T细胞作用的机理进行探讨,根据已发表的SEA、SEB和SEC1基因全序列,用T细胞抗原表位预测软件Guotif2.0对其进行T细胞抗原表位预测,统计与HLAⅠ和HLAⅡ各抗原位点结合的SEA、SEB和SEC1肽段的出现次数。结果显示,SEA、SEBT SEC1具有共同的特点,即都是主要与HLAⅠ类分子的A3位点和HLAⅡ类分子的DR1位点具有较强的结合。说明SEA、SEB和SEC1与HLAⅠ类分子和HLAⅡ类分子都有很强的结合性。三者在HLAⅠ和HLAⅡ结合位点上具有较强的同源性。本研究为SE活化T细胞作用机制的功能实验提供了依据。     

GGTA1~(-/-)五指山小型猪SLAI类基因分子特征及与HLA相似性分析

目的明晰α-1,3半乳糖基转移酶基因敲除(GGTA1-/-)的五指山小型猪SLA经典I类基因分子结构特征及其与人HLA的相似性,对研究异种器官移植细胞性排斥反应具有重要意义。方法采集6头祖代GGTA1-/-五指山小型猪耳组织,利用RT-PCR对SLA I类基因(SLA-1、SLA-3、SLA-2)扩增、克隆及测序,对序列进行BLAST分析,并采用生物信息学方法对SLA I类基因分子结构特征及其与人HLA的同源性进行分析。结果测序分析表明,共获得6条等位基因序列,其中4条为已公布的等位基因(SLA-1*0703、SLA-2*1102、SLA-3*0401、SLA-3*0403),另2条为新等位基因(SLA-1*0401wz01、SLA-2*11wz01)。五指山小型猪与人HLA同源性为70.5%~72.1%。SLA-1*0401wz01、SLA-1*0703、SLA-2*11wz01、SLA-2*1102和SLA-3*0401的CD8+分子识别的关键结合域与人HLA氨基酸序列比对,均仅在位点225、228处发生了突变(T→S、T→M),其他位点高度保守。SLA-2*11wz01和SLA-2*1102与人HLA I类基因NK细胞抑制性受体(killer inhibitory receptor,KIR)结合区氨基酸同源性较高,在NKTA-1亚型结合域内仅有1个氨基酸差异,而在NKTA-2和NKTA-3亚型结合域内有2个氨基酸差异。结论从免疫细胞介导的异种排斥反应角度出发,GGTA1-/-五指山小型猪SLA I类基因氨基酸序列与人HLA高度相似,可作为未来猪-人异种器官移植的良好供体之一。     

CXCL12-α基因编码区的克隆与生物信息学分析

目的:克隆人源CXCL12-α的成熟肽编码序列后进行生物信息学分析及其蛋白质结构与功能预测。方法:采用RTPCR方法从人骨髓组织中克隆CXCL12-α基因序列,并将编码其成熟蛋白的核酸片段插入原核表达载体pET-30a(+)中,转化Escherichia coli后进行酶切鉴定和DNA序列测定。利用在线网络生物信息学相关数据库和分析软件对测序结果进行分析,并对重组蛋白的一、二、三级结构及功能等进行验证和预测。结果:获得了基因序列为263 bp的人源CXCL12-α基因,与GenBank中公布序列一致。双酶切鉴定及DNA测序验证结果正确。生物信息学检索该基因编码蛋白的氨基酸序列与理化参数显示,蛋白无跨膜区,在第29位有一个Ser为蛋白激酶磷酸化位点,第1~21位可能为信号肽区域。ɑ-螺旋,无规则卷曲,延伸链和β-转角数量分别占总二级结构的44.94%、22.47%、21.35%、11.24%。同源建模预测信息可信度为0.68,该蛋白G-factor结构计分总平均为0.33,结构检验表明该蛋白属于正常范围内。二级结构和拓扑结构信息、蛋白质结合位点三维图和预测蛋白可能催化口袋及结合位点、三维结构模拟的可视化结构显示其空间结构稳定。结论:人源CXCL12-α分子克隆成功,网络数据库等生物信息学分析及结合蛋白质结构与功能预测可为深入研究CXCL12-α提供理论指导。     

重型β-地中海贫血异基因造血干细胞移植术后的护理

目的总结异基因造血干细胞移植治疗重型β-地中海贫血的护理要点。方法对2007年12月~2014年2月在我科行异基因造血干细胞移植治疗的112例重型β-地中海贫血患儿的临床资料进行回顾性分析。结果 96例从层流病房转入普通病房治疗的患儿通过环境保护、饮食护理、心理护理等,有效地减少了并发症的发生,为家庭护理管理起到了承上启下的作用。结论精心的护理可降低儿童造血干细胞移植术后并发症发生率,促进了患儿的康复。     

贵州白山羊GOLA-DQA1基因多态性及生物信息学分析

目的:通过筛选贵州白山羊GOLA-DQA1基因SNPs位点,为进一步研究MHC基因多态性与山羊免疫性状的相关性提供依据。方法:选取同一生长环境中的贵州白山羊母羊157只构建品种DNA池并进行测序,结合SNPs位点测序峰高比值估算等位基因频率,利用软件对不同基因型的RNA和蛋白质二级结构进行预测。结果:在贵州白山羊DQA1基因发现6个SNPs位点G+2930A(同义突变)、T+2959C(Asn→Ser)、G+3061A(Pro→Leu)、A+3082G(Me→Thr)、C+3463A(Trp→Cys)、C+3525T(Arg→His)。生物信息学分析发现,C+3463A变异位点使RNA二级结构更加稳定;A+3082G变异导致自由能增加,稳定性降低。蛋白质结构预测发现除A+3082G基因型与原序列具有相同的二级结构外,其他基因型均引起蛋白质二级结构的改变。结论:贵州白山羊GOLA-DQA1基因具有丰富的多态性,但SNPs位点等位基因频率差异较大。     

环江香猪FSHβ、ESR及ZAR1基因多态性分析

环江香猪是广西著名的地方品种,本研究采用PCR-RFLP、HRM结合测序的方法分析了广西环江香猪繁殖状相关基因FSHβ、ESR和ZAR1基因的多态性。结果显示,首次成功克隆测序了环江香猪ZAR1基因的外显子3,与Gen Bank中的ZAR1基因(DQ231443,gi:83727928)外显子3序列100%同源。针对134头环江香猪检测样本中均未发现FSHβ和ESR基因的多态突变位点。对ZAR1基因的外显子3和部分内含子3测序后发现5个新突变SNP位点,未见有已报道的对猪产仔数产生显著影响的外显子3(C54T)突变。论文显示不同品种猪基因突变多态性亦呈现为不同的模式,研究结果对建立环江香猪基因标记辅助选择方法具有重要意义。     

猪SLA-DR二级结构预测及同源模建

应用EXPASY服务器（http：／／www．expasy．oh／tools）上的SOPMA法对SLA—DR的α链和口链进行二级结构的预测,并与人的HLA—DR相应的α链和β链的氨基酸和二级结构成分比较,在此基础上同源模建SLA—DRα链、β链及复合体SLA—DR的三级结构。结果显示,SLA—DR的α链各二级结构成分α螺旋、β折叠、转角和无规则卷曲的数目分别为36、61、4和69,β链中分别为42、56、16和74。α链和β链各二级结构成分与复合体SLA—DR具有高度的符合率,分别达到98．7％（α螺旋）、99．1％（β折叠）、83．3％（转角）和97．2％（无规则卷曲）。各功能区分析,SLA—DR的α1和β1区为结合抗原的高变化区。同源模建和三级结构分析表明SLA—DR的α链和口链具有独立的三级结构,并可以组成复合体SLA—DR。     

The HLA molecules DQA1*0501/B1*0201 and DQA1*0301/B1*0302 share an extensive overlap in peptide binding specificity

Assays to measure the binding capacity of peptides for HLA-DQA1*0501/B*0201 (DQ2.3) and DQA1*0301/B*0302 (DQ3.2) were developed using solubilized MHC molecules purified from EBV-transformed cell lines. These quantitative assays, based on the principle of the inhibition of binding of a high-affinity radiolabeled ligand, were validated by examining the binding capacity of known DQ-restricted epitopes or ligands. The availability of these assays allowed an investigation of patterns of cross-reactivity between different DQ molecules and with various common DR molecules. DQ2.3 and DQ3.2 were found to have significantly overlapping peptide binding repertoires. Specifically, of 13 peptides that bound either DQ2.3 or DQ3.2, nine (69.2%) bound both. The molecular basis of this high degree of cross-reactivity was further investigated with panels of single substitution analogs of the thyroid peroxidase 632-645Y epitope. It was found that DQ2.3 and DQ3.2 bind the same ligands by using similar anchor residues but different registers. These data suggest that in analogy to what was previously described for HLA-DR molecules, HLA-DQ supertypes characterized by largely overlapping binding repertoires can be defined. In light of the known linkage of both HLA-DQ2.3 and -DQ3.2 with insulin-dependent diabetes mellitus and celiac disease, these results might have important implications for understanding HLA class II autoimmune disease associations.     

A newHLA-DRB1 * 13 allele (DRB1 * 1318) with a shortDRB1*08 sequence

The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession number Z48631. The name listed for this sequence was officially assigned by the WHO Nomenclature Committee in November 1994. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1994), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report     

CYP1A1*2 and CYP1A1*3 polymorphisms cosegregate in the Indian population

Several ethnic groups have been genotyped for polymorphisms at the CYP1A1 gene locus that encodes the enzyme that catalyzes the initial step in the metabolism of polycyclic aromatic hydrocarbons. Two of the CYP1A1 polymorphisms, namely, CYP1A1*2 and CYP1A1*3 are reported to cosegregate among the Japanese and to a lesser extent in Caucasians, but not in people of African descent. In the absence of such information in the Indian population, the frequency of the CYP1A1*2 polymorphism was determined in this study, using DNA samples from 649 ethnic Indians who had been earlier genotyped for the CYP1A1*3 polymorphism. Analysis of the combined genotype data revealed that the two polymorphisms cosegregate in the Indian population.     

A new DRB1 * 1202 allele (DRB1 * 12022) found in association with DQA1 * 0102 and DQB1 * 0602 in two Black narcoleptic subjects

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number L34353. The name listed for this sequence has been officially assigned by the WHO Nomenclature Committee in August 1994. This follows the agreed policy that subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al 1994), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report     

Human orosomucoid polymorphism: molecular basis of the three common ORM1 alleles, ORM1*F1, ORM1*F2, and ORM1*S

The human orosomucoid (ORM) is controlled by two closely linked loci, ORM1 and ORM2, and two tandem genes, AGP1 and AGP2, encoding the proteins produced by the two loci, have been cloned. In this study the molecular basis of ORM1 polymorphism was investigated. For the detection of mutations the products of the six exons of each gene, amplified by the polymerase chain reaction (PCR), were screened by single-strand conformation polymorphism analysis. Subsequently, the exons with an altered migration pattern were gene-specifically amplified by nested PCR. Sequencing of the gene-specific PCR products showed that the three common ORM1 alleles result from A→G transitions at the codons for amino acid positions 20 in exon 1 and 156 in exon 5 of the AGP1 gene: ORM1*F1 was characterized by CAG (Gln) and GTG (Val), ORM1*F2, by CAG (Gln) and ATG (Met), and ORM1*S, by CGG (Arg) and GTG (Val). The phylogenesis of the genes encoding these three ORM1 alleles is discussed. Received: 5 September 1996     

Comparison of acid phosphatase ACP1 variants by isoelectric focusing and conventional electrophoresis: identification of three new alleles, ACP1*N, ACP1*P and ACP1*S

Three new alleles of human red cell acid phosphatase (ACP1) have been identified by comparison with previously reported variants using three different electrophoretic techniques. Family data are available on all the variants and show genetic transmission of the rare alleles ACP1*N, ACP1*P and ACP1*S. Further evidence of a rare allele demonstrating reversed 'A' activity is also described. The report documents the need to use several electrophoretic techniques to characterize new or rare variants.     

AM1* parameters for bromine and iodine

Our extension of the AM1 semiempirical molecular orbital technique, AM1*, has been parameterized for the elements Br and I. The basis sets for both halogens contain a set of d-orbitals as polarization functions. AM1* performs as well as other MNDO-like methods that use d-orbitals in the basis, and better than those that rely on an sp-basis. Thus, AM1* parameters are now available for H, C, N, O and F (which use the original AM1 parameters), Al, Si, P, S, Cl, Ti, Cu, Zn, Br, Zr, Mo and I. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.