KIR2DL4-IgFc融合蛋白基因真核细胞表达载体的克隆及鉴定分析

目的：构建人KIR2DL4－IgFc段融合蛋白基因真核细胞表达载体。方法：用RT－PCR从孕妇蜕膜组织单个核细胞的总mRNA中逆转录扩增KIR2DL4细胞外段cDNA，经Nhe I和BamH I双酶切后，定向插入真核细胞表达载体CD5lnegl中，然后经酶切和测序鉴定。结果：限制性内切酶切酶 切和序列分析表面已成功构建CD5lnegl-KIR2DL4载体，结论：本研究成功构建KIR2DL4－IgFc融合蛋白真核细胞表达载体，为研究KIR2DL4与其配体之间的关系奠定了基础。     

KIR2DL5基因研究进展

杀伤细胞免疫球蛋白样受体(killer-cell immunoglobulin-like receptors,KIPs)是由一组位于19q13.4的紧密成簇的基因编码的免疫球蛋白超家族受体,蛋白结构具有多样性。KIR2DL5由于其独特的基因转录表达特征、单倍体分布、群体分布和表达的多样性,以及D0-D2免疫球蛋白样胞外段结构域和较长的胞内段结构特征而有别于其他KIR,在KIR研究中尤为重要。本文拟对这一基因的研究进展作一综述。     

KIR2DL4在NK-92细胞上的超表达及其功能分析

目的 克隆KIR2DL4 cDNA，并使其在NK-92细胞上获得稳定表达，分析KIR2DL4分子对NK-92细胞功能的调节作用。方法 采用RT-PCR方法，从人蜕膜单个核细胞扩增出KIR2DL4 cDNA，将目的基因亚克隆于逆转录病毒表达载体构建成KIR2DL4-pLNCX表达载体，将重组质粒转入NK-92细胞，利用单克隆抗体#33进行FACS检测，观察KIR2DL4分子在靶细胞表面的表达。ELISA检测KIR2DL4对IFN-γ分泌的影响，同时根据LDH的释放实验，分析KIR2DL4分子对NK-92细胞毒功能的调节。结果：KIR2DL4分子在经KIR2DL4-pLNCX转染的靶细胞表面获得稳定高表达，且不影响其他受体在：NK-92细胞上的表达水平。KIR2DL4分子能够部分限制：NK-92细胞对HLA-G阳性靶细胞的杀伤作用，交联KIR2DL4分子能够诱导IFN-γ的分泌。结论 成功构建了KIR2DL4-pLNCX逆转录病毒表达载体，并使KIR2DL4分子在NK-92细胞上获得功能性的高表达。     

KIR2DL分子的研究进展

KIR2DL分子是NK细胞受体(NKR)的一种,属免疫球蛋白样受体家族成员,分布在NK细胞和部分细胞毒性T淋巴细胞上。KIR2DL与相应的配体HLA-C结合后传递抑制信号,使NK细胞对靶细胞的杀伤作用被抑制。本文对此受体的结构及其识别与信号转导机制的研究进展作一综述。     

KIR3DL1基因启动子亚克隆及表达调控载体的构建

为研究KIR3DL1基因的表达调控机制,亚克隆KIR3DL1基因的核心启动子序列,并构建KIR3DL1基因启动子-荧光素酶报告载体,采用PCR法从含有KIR3DL1基因转录起始位点5'侧翼区的质粒中扩增KIR3DL1基因核心启动子序列,产物纯化回收后与pGEM-T easy载体连接,测序鉴定正确的重组子经限制性内切酶BglⅡ和NcoⅠ双酶切后,插入同样经BglⅡ和NcoⅠ双酶切的用于基因表达调控研究的pGL3-Basic报告载体,最终获得了长度为254bp的KIR3DL1基因启动子克隆,构建了调控荧光素酶报告基因的真核表达载体.通过酶切及基因测序的方法证实所构建的重组子是正确的,说明KIR3DL1基因启动子表达调控载体的构建是成功的.     

NK细胞受体KIR2DL4的结构与功能

KIR2DL4分子是NK细胞受体(NKR)的一种,属于免疫球蛋白样(IgSF)受体家族成员,主要分布在自然杀伤(Natural killer,NK)细胞上。KIR2DL4是HLA-G分子的特异性受体,结构上具备激活性和抑制性受体的双重特点,能够通过不同途径影响NK细胞的活性,具有重要的免疫调节功能。     

HLA-Gα1结构域Met76和Gln79在KIR2DL4特异性识别中的作用研究

目的 探讨HLA-G α1结构域中特异的Met76和Gln79两个位点在HLA-G特异性受体KIR2DL4识别中的作用.方法 采用真核表达载体CD51neg1,克隆表达KIR2DL4胞外区与IgGFc段的可溶性融合蛋白;利用“桥式”PCR和“点突变”方法,将位于HLA-G目的基因α1结构域中编码Met76和Gln79的密码子突变为Ala76,79（HLA-mG）,通过逆转录病毒表达载体分别使野生型HLA-G及其突变体在HLAⅠ分子阴性的K562细胞上表达.流式细胞术分别测定KIR2DL4-IgG Fc融合蛋白与野生型HLA-G及Met76、Gln79→Ala76,79 HLA-G突变体结合的荧光强度,通过比较两者的平均荧光强度分析HLA-G分子Met76、Gln79残基在HLA-G与其受体KIR2DL4识别过程中的作用.结果 Western blot结果显示,本实验成功表达KIR2DL4-IgG Fc融合蛋白.FACS结果表明,野生型HLA-G及Met76、Gln79→Ala76,79 HLA-G突变体在K562细胞上高表达.Met76、Gln79突变为Ala76,79后能显著影响KIR2DL4对HLA-G分子的识别.结论 HLA-Gα1结构域中Met76和Gln79可能是其特异性受体KIR2DL4识别的关键位点.     

上海地区汉族人群KIR2DL5基因多态性及单元型分析

KIR2DL5基因是杀伤细胞免疫球蛋白样受体 (KIR )单元型B的特征性标志 ,至少有 4个变异体 ,其中 2DL5A 0 0 1和2DL5B 0 0 3是表达型 ,统称为KIR2DL5 1;2DL5B 0 0 2和 2DL5B 0 0 4是不表达型 ,统称为KIR2DL5 2。本文用序列特异性引物PCR法 (PCR SSP )分析了KIR2DL5表达型KIR2DL5 1和不表达型KIR2DL5 2在上海地区正常汉族人群的分布及其在KIR单元型的组成。结果发现 (1)在 87名健康汉族人和 14个家族中表达型KIR2DL5 1和不表达型KIR2DL5 2基因频率分别为0 1977和 0 0 4 11;(2 )KIR2DL5基因变异体在不同单元型中分布不同 ,有 5种不同组合 ,其中单元型 1、 2、 3、 4、 11、 12、15和 16不含KIR2DL5及其变异体 ;单元型 5和 14只含有KIR2DL5 1;单元型 13和 17只含有KIR2DL5 2 ;单元型 6含有单一KIR2DL5 1或二者兼有 ;单元型 8和 9同时含有KIR2DL5 1和KIR2DL5 2。     

浙江汉族人群 KIR3DL2等位基因的分布

目的:分析浙江汉族人群 KIR3DL2等位基因分布。 方法:基因组DNA提取采用磁珠法。应用PCR技术通过4对引物扩增 KIR3DL2基因的全长序列,利用BigDye terminator v3.1测序试剂盒对208名无血缘关系的汉族无偿献血者的全部外显子序列进行测序分析。根据 ...     

KIR2DL4基因的多态性与南方汉族白血病的相关性研究CSCD

目的探讨南方汉族人群白血病与KIR2DL4等位基因多态性的相关性。方法提取南方汉族急性淋巴细胞白血病（acute lymphoblastic leukemia,ALL）（n=283）、急性髓系白血病（acutemyeloid[eukemia,AML）（n=227）、慢性髓系白血病（chronic myeloid leukemia,CMI。）（n=80）患者和306名南方汉族随机正常志愿捐血者的DNA基因组,应用本实验室自行设计的引物对KIR2DL4基因的全部8个外显子进行测序分型,用Assign4．7分析软件判定基因型。用SPSS13．0统计软件分别对检出的每个KIR2DIA等位基因、10A型等位基因和9A型等位基因的检出比例分别进行差异显著性分析,并进行P值校准（Pc）。结果ALL、AML、CMI,患者和正常对照均检出5种等位基因：KIR2DIA＊001、＊005、＊006、＊008、＊011。ALL病例组与对照组的KIR2DI．4＊011等位基因检出比例、10A型KIR2DL4等位基因检出比例的差异有统计学意义（KIR2DL4＊011：OR=1．66,P=0．01;10A型K琥2DIA：OR=0．42,P=0．03）,但尸值经过校准后,差异均无统计学意义（Pc≥O．05）。AML、CML病例组分别与对照组检出的各个KIR2DL4等位基因、10A型等位基因和9A型等位基因的检出比例进行比较,组间比较差异均无统计学意义（P〉0．05、Pc〉0．05）。结论南方汉族ALL、AML、CML白血病与KIR2DL4等位基因多态性无显著关联。     

Possible roles of KIR2DL4 expression on uNK cells in human pregnancy

PROBLEM: To investigate possible roles of the natural killer (NK) cell receptor killer immunoglobulin-like receptor (KIR)2DL4 expressed on uterine NK (uNK) cells during pregnancy, we investigated KIR2DL4 expression on uNK cells isolated from patients with early recurrent spontaneous abortion (RSA) and normal early pregnancy women, and functions of KIR2DL4 was analyzed in vitro. METHODS OF THE STUDY: Semi-quantitative RT-PCR analysis was introduced to detect KIR2DL4 messenger RNA (mRNA) expression on uNK cells. Cytotoxicity and cytokine production as the result of interaction of KIR2DL4 and its ligand human leukocyte antigen (HLA)-G were analyzed in vitro with lactic dehydrogenase releasing method and enzyme-linked immunosorbent assay, respectively. RESULTS: No significant difference in KIR2DL4 mRNA expression was observed, while the KIR2DL4 protein level in isolated uNK cells is much higher in normal controls than that in RSA patients. Data showed that HLA-G transfection could not reverse the lysis of uNK against HLA-G transfected K562 cells but induced cytokine production. Furthermore, we demonstrated that, via KIR2DL4, membrane-bound HLA-G could induce high cytotoxicity and cytokine production in a high cytotoxic, IL-2 dependent human NK cell line NK-92 cells. CONCLUSION: Our data suggest that KIR2DL4 might play a crucial implication for human pregnancy.     

No association of KIR3DL1 or KIR3DS1 or their alleles with ankylosing spondylitis

Abstract
We determined the alleles of KIR3DL1 and KIR3DS1 in a cohort of British Caucasian ankylosing spondylitis (AS) patients and HLA-B27-positive controls. We found no association in frequencies of the alleles of these genes in AS. In addition, no differences were found when the patients and controls were differentiated by gender.     

Investigation of killer cell immunoglobulin-like receptors gene KIR3DL2 diversity and confirmation of KIR3DL2*015 in a Chinese population

Human killer cell immunoglobulin-like receptors (KIRs) are a subfamily of the immunoglobulin superfamily. Here, we established polymerase chain reaction-sequence-based typing (PCR-SBT) procedure to identify alleles of KIR3DL2 gene. The method was designed around the specific amplification of exon 3 to exon 4 and exon 8 to exon 9 of the KIR3DL2 gene. Genomic DNA from 104 healthy, unrelated Chinese Han individuals was typed for KIR3DL2 alleles. Each sample was assigned to the putative allele combination according to the sequences of all KIR3DL2 alleles. We observed 18 different genotypes and eight KIR3DL2 alleles in the population, with KIR3DL2*002 having the highest frequency of 0.558, and confirmed the new KIR3DL2*015 allele. Our data showed that the established PCR-SBT methods for KIR3DL2 allele typing were reliable, and Chinese Han population is distinct in KIR3DL2 allele frequencies.     

KIR2DL1 allelic diversity: four new alleles characterized in a bone marrow transplant population and three families

Sequencing of PCR amplified genomic DNA including most of the coding region was used to identify killer cell immunoglobulin-like receptor 2DL1 alleles from three families and 77 bone marrow transplant patients and donors. Alleles 2DL1*00302 and *002 were frequently observed in addition to two other known alleles and four new alleles, 2DL1*00402, 2DL1*007, 2DL1*008, and 2DL1*009.     

Investigation of killer cell immunoglobulin-like receptor gene diversity III. KIR2DL3

The allelic variation of one of the chromosome 19 KIR genes, KIR2DL3, has been investigated using a polymerase chain reaction sequence-specific oligonucleotide probe-based methodology. The procedure has been applied to a healthy Northern Irish control group in order to establish phenotype and genotype frequencies in this Caucasian population. In addition, cell line DNA and Centre d'Etude du Humaine (CEPH) families, both from the 13th International Histocompatibility Workshop have been investigated, establishing control data for this gene.     

Investigation of killer cell immunoglobulin-like receptor gene diversity V. KIR3DL2

Allelic definition within the killer cell immunoglobulin-like receptor gene, KIR3DL2, has been achieved through a sequence-specific oligonucleotide probe methodology, designed around the specific amplification of the D0 and D1 domains and a section of the cytoplasmic tail of this gene. The system has been applied to a healthy Northern Irish control group, establishing frequencies for this Caucasian population. Additionally, the KIR3DL2 allele status of cell line DNA and Centre d'Etude du Polymorphisme Humain (CEPH) families, both from the 13th International Histocompatibility Workshop, has been established. A high level of KIR3DL2 allelic polymorphism has been identified.