GAD65基因特异siRNA的构建和功能检测

目的构建GAD65基因特异的siRNA并检测体外功能。方法应用RT-PCR方法克隆大鼠GAD65基因、测序、重组病毒包装检测体外功能。针对GAD65基因的编码区,设计3条siRNA片段及一条对照片段,将其分别亚克隆于siRNA的慢病毒表达载体并小量包装获得病毒颗粒(LV-GFP-siRNA-r GAD65);用siRNA病毒颗粒感染GAD65过表达的293细胞,检测GAD65表达情况,从中筛选出高效特异的siRNA。大量包装特异的siRNA病毒颗粒,在体外感染培养的大脑皮质细胞,在体内注射到SD大鼠的苍白球内侧部,检测r GAD65的表达水平,观察siRNA的功能。结果应用RT-PCR方法克隆r GAD65基因,其序列与Gen Bank中的基因序列几乎一致(99.72%),包装病毒后在293细胞中过表达。筛选出针对r GAD65的高效特异的siRNA,在GAD65基因的沉默效率可达到66%。在体外、体内分别应用Western印迹检测感染了LV-GFP-siRNA3-r GAD65的大脑皮质细胞和定点注射了LV-GFP-siRNA3-r GAD65的大鼠GPi。结论成功克隆r GAD65基因,设计、筛选出高效特异的siRNA,并证明这种siRNA在体内和体外均能有效地沉默r GAD65的表达。     

两种GAD65片段与IL-4基因共表达DNA疫苗的构建与鉴定

目的构建两种谷氨酸脱羧酶（GAD）65片段与白细胞介素4（IL-4）基因共表达DNA疫苗,并在体外COS-7细胞中对表达产物进行检测。方法从GAD65质粒中扩增出GAD190～315和GAD490～570两个片段的cDNA,Overlap法分别与hIL-2信号肽cDNA基因拼接,将拼接后的融合基因与IL-4基因依次克隆入双启动子真核表达载体pBudCE4.1中。重组子经酶切和测序鉴定后,脂质体转染COS-7细胞,Western blot法检测融合基因的表达,ELISA法检测IL-4表达。结果核酸序列测定表明克隆的融合基因和IL-4基因序列与报告序列一致,开放阅读框正确,Western blot和ELISA显示在COS-7细胞中均可检测两个目的基因的表达。结论两种GAD65片段与IL-4共表达DNA疫苗均成功构建,为1型糖尿病的预防研究提供了实验基础。     

糖尿病免疫学新进展

I型糖尿病是儿童和成人的常见慢性病，环境因素是作为启动因素还是加速因素尚不清楚。最近10年的糖尿病研究表明，I型糖尿病的发生与胰岛自身免疫密切相关，它是一种T一细胞介导的胰岛自身免疫性疾病。目前还没有一个研究β细胞自身活化T细胞的方法，胰岛炎无创性检查亦不可能。但近年胰岛自身抗原的分子克隆（如GAD、ICA）为研究胰岛自身免疫提供了很大帮助。1981年有人描述在1型糖尿病病人体内有一种64K的自身抗原，1990年被确认为谷氨酸脱竣酶（GAD），1991年克隆出了新的GAD异构体（GAD65）。64K蛋白胰蛋白酶片断40K和37K也分…     

1型糖尿病关键抗原谷氨酸脱羧酶65蛋白表达的研究现状

谷氨酸脱羧酶（GAD）属于内分泌酶,包括GAD65和GAD67两种同工酶,但只有GAD65才是重要的胰岛自身抗原.众多研究认为GADA是诊断和预测1型糖尿病（T1DM）和成人隐匿性自身免疫糖尿病（LADA）最敏感且应用最广泛的指标[1-2].GADA检测方法多样,包括放射配体法和酶联免疫吸附实验（ELISA）法等,但诊断敏感性和特异性与GAD65蛋白纯度和抗原性密切相关.而抗原特异性T细胞更能直接反映胰岛自身免疫状态,可改善抗体诊断T1 DM的效率.研究表明在新诊断T1DM患者中使用酪氨酸磷酸酶（IA-2）、GAD65、胰岛细胞特异性葡萄糖-6-磷酸酶催化亚基相关蛋白（IGRP）和前胰岛素原作为CD8＋T细胞刺激抗原,其诊断敏感性波动在25％～40％,特异性在87％ ～ 100％[3].随后T细胞检测国际标准化工作组证实GAD270-285是人白细胞抗原（HLA）Ⅱ类分子四聚体检测技术的主要刺激片段[4-5].因此,选用合适的GAD65抗原或蛋白片段可改善GADA或自身反应性T细胞的诊断敏感性和特异性.     

细粒棘球绦虫egG1Y162抗原基因的克隆及序列分析

根据多房棘球绦虫emY162基因序列设计引物,利用PCR以细粒棘球绦虫原头蚴和成虫的基因组DNA和cDNA为模板扩增egG1Y162基因,构建PUCm-T/egG1Y162和PUCm-T/egG1Y162 cDNA重组质粒,经PCR、酶切及测序后,进行序列分析。细粒棘球绦虫2个不同发育阶段均克隆出egG1Y162基因,从基因组DNA和cDNA克隆得到的片段长度分别为1 680 bp和459 bp,登录号分别为AB458258和AB458259。     

人重组谷氨酸脱羧酶65基因的克隆表达及其在1型糖尿病诊断中的初步应用

目的 构建人重组谷氨酸脱羧酶65(GAD65)基因不同区段原核表达载体,诱导表达获得重组蛋白,并初步验证GAD65不同抗原区段在1型糖尿病GAD自身抗体检测中的价值.方法 应用巢式逆转录聚合酶链式反应(RT-PCR)技术调取目的 基因,构建相应的原核表达质粒,转化大肠杆菌E.coli HB101,诱导表达获得纯化重组蛋白,用重组蛋白作为包被抗原,初步建立检测GAD自身抗体的酶联免疫吸附测定法(ELISA)方法,评价各片段在1型糖尿病诊断中的价值.结果 获得了4种可被1型糖尿病患者血清识别的重组人GAD抗原区段,其中GAD65(180-585)抗原区段具有很好的特异性,检出率为55.3%,是首选的抗原区段.结论 所选重组人GAD65(180-585)抗原区段具有良好的抗原性,可作为1型糖尿病患者辅助诊断试剂的候选抗原.     

剔除护骨素基因的小鼠胚胎干细胞杂合子模型的建立

目的建立护骨素（OPG）基因剔除小鼠胚胎干细胞杂合子模型，为建立用于研发抗骨质疏松药物的基因剔除动物模型奠定基础。方法PCR方法扩增两条同源臂，将两条用于同源重组的片段插入XpPNT载体，构建OPG基因敲除打靶载体。线性化及纯化以后通过电穿孔转导小鼠胚胎干细胞，并用G418和Gancyclovir进行双药筛选培养，得到双药抗性克隆后抽提基因组DNA，采用PCR和southern杂交方法确定同源重组的胚胎干细胞克隆。结果经双药筛选得到124个双药抗性克隆，PCR和southern杂交鉴定获得一个发生同源重组的胚胎干细胞克隆。结论总之，本研究成功获得了OPG（-／＋）杂合子小鼠胚胎干细胞克隆，为进一步通过显微注射及交配育种获得OPG基因剔除小鼠，在活体水平研究OPG基因的功能打下基础，同时也使建立用于研发抗骨质疏松药物的基因剔除动物模型成为可能。     

055 改变胰岛β细胞表面GAD的表达可控制NOD小鼠自身免疫性糖尿病的发病

[英]/Yoon JW…//Science. -1999,5417.-1183～1187 本实验利用转基因小鼠,研究了β胰岛细胞上谷氨酸脱羧酶(GAD)的表达与非肥胖糖尿病(NOD)小鼠糖尿病发病的确切关系。 将两种不同变异型的小鼠GAD cDNA(rGAD65和rGAD67)的反义基因转移到NOD小鼠的胚系细胞上,然后将这些干细胞植入雌性NOD鼠,经传代得到六个表达GAD反义基因的转基因NOD小鼠品系。将此六系小鼠分为两组,第1组小鼠根据它们表达的转移基因数目的相对多少记为H-AS-GAD-NOD系、M-AS-GAD-NOD系和L-AS-GAD-NOD系;第二组中的三系小鼠依同样的标准记为HK-AS-GAD-NOD系、MK-AS-GAD-NOD系和LK-AS-GAD-NOD系;对照组是相应数量的不表达GAD反义基因的NOD小鼠(Transgenic Negative,TN组)。蛋白免疫印渍分析结果表明：H-AS-GAD-NOD系小鼠完全抑制了GAD在β胰岛细胞上的表达,M-AS-GAD-NOD和L-AS-GAD-NOD则中度和轻度抑制了GAD的表达,而GAD在上述各实验组小鼠的脑组织中的表达却没有显著差别。 研究还监测了各系转基因NOD小鼠与TN组小鼠的糖尿病发病情况,发现第40周时,第1组15例H-AS-GAD-NOD系小鼠没有1例发病,而M-AS-GAD-NOD系、L-AS-GAD-NOD系和TN小组小鼠的发病率分别为67％(12 of 18)、75％(12 of 16)和81％(17 of 21)。第20周时组织学检查显示：H-AS-GAD-NOD系80％以上的胰岛未受损伤,患有胰岛周炎者少于20％;其它系和对照TN组的大部分小鼠患中度或严重的胰岛炎。第2组结果与第1组相似。作为转基因的对照,本研究将另一种细胞表面的自身抗原--内源性逆转录病毒衣壳蛋白的反义基因转移到NOD小鼠的染色体DNA上,发现即使这些小鼠完全表达了该转移基因,其糖尿病的发病率仍接近没有表达该基因的NOD小鼠(82％)。上述结果表明,NOD小鼠GAD的特异性缺失可防止NOD小鼠糖尿病的发生。 将20周雌性H-AS-GAD-NOD系小鼠的脾细胞输入到6～8周的联合T细胞缺陷的NOD小鼠(NOD.scid小鼠)体内,10周以后,8例NOD.scid小鼠没有1例发病;而将TN组小鼠的脾细胞输入到另外10只NOD.scid小鼠体内,10周后则有9例小鼠患了糖尿病。实验结果表明表达GAD反义基因的NOD小鼠体内GAD特异性的T细胞的增殖受到了抑制。 结论 研究认为GAD是GAD特异的T细胞引发NOD小鼠1型糖尿病的关键抗原,β胰岛细胞GAD的表达缺失可保护细胞免受T细胞介导的免疫攻击,可防止NOD小鼠1型糖尿病的发生。 (沈文浩摘 白 云校)     

重型乙型肝炎患者血清乙型肝炎病毒全基因克隆及测序

目的建立重型乙型肝炎患者血清乙型肝炎病毒（HBV）DNA克隆并测序，从全基因水平分析HBV基因变异与重型乙型肝炎发病的关系。方法10例重型乙型肝炎患者血清提取HBV DNA，聚合酶链反应（PCR）扩增HBV全基因。PCR产物构建到PUCm-T载体上，转化至大肠杆菌感受态DH-5α细胞，经酶切鉴定，获得含3．2Kb HBVDNA的重组克隆菌，全基因测序，分析各读码框核苷酸和氨基酸变化。结果4例成功构建HBV DNA克隆，并完成全基因测序。其中3例在前C区发生G1896A变异，产生一个终止密码子，导致HBeAg缺失；1例在C启动子区1762、1764双位点出现突变；有多处点突变及缺失变异分布于PreS2区及C区已知细胞毒T淋巴细胞、B淋巴细胞和T淋巴细胞的细胞表位。结论该法可用于临床研究HBV病毒基因结构与重型乙型肝炎发病的关系，并为进一步研究其HBV基因功能奠定基础。     

111胰岛细胞自身抗体初期阳性的儿童出现Ⅰ型糖尿病临床症状后的β细胞功能恶化

医生新近诊断的Ⅰ型糖尿病患儿大多数可发现有胰岛细胞特异性抗体。胰岛细胞抗体（ICA）的发生率为cd％－86％，而胰岛素自身抗体（LAA）则为18％－69％；同时在初诊时发现70％－80％的患者对谷氨酸脱竣酶的65kDa同功酶（GAD65A）产生抗体。糖尿病表现症状以后，ICA和残余B细胞功能之间的关系仍有不少争议。为了研究ICA、IAA和GAD65A的起始阳性表现对糖尿病酮症酸中毒（DKA）的严重性以及对残余p细胞功能的影响，本文作者对芬兰的780名儿童和青少年（年龄0．8－14．9岁）在作出1型糖尿病诊断时，对其ICA、IAA和GAD65A状况进…     

Molecular cloning and amino acid sequence of brain L-glutamate decarboxylase.

We used specific polyclonal antibodies against L-glutamate decarboxylase (GAD) to screen a mouse brain cDNA library that was constructed in the expression vector lambda gt11. We obtained 1.5 x 10(6) recombinant DNA clones in the mouse brain cDNA library. One of the clones was positively identified as a GAD clone on the basis of the following results: (i) the clone and its secondary and tertiary clones all reacted strongly with anti-GAD antibodies; (ii) the fusion protein obtained from lambda GAD-Y1089 showed good GAD enzyme activity as determined by both CO2 and gamma-aminobutyric acid methods. The GAD clone thus obtained contains GAD cDNA of approximately 2.6 kilobases that has one internal EcoRI site. After GAD cDNA was cut at the EcoRI site, two DNA fragments of about 1.6 and 1.0 kilobases were obtained at the 5' and 3' ends, respectively. The cDNA insert was found to be composed of 2632 base pairs, the translation initiation site was assigned to the methionine codon ATG, and the termination site was found to be TGA (positions 2216-2218). Furthermore, the coding region in 2169 base pairs was found to consist of 723 amino acids. The protein has a molecular weight of 83,207 and contains 83 strongly basic, 108 strongly acidic, 226 hydrophobic, and 221 polar amino acids with an isoelectric point of 5.355. The relationship of this GAD cDNA to other forms of GAD is discussed.     

细粒棘球蚴egG1Y162抗原基因的克隆及蛋白质序列分析

目的克隆细粒棘球蚴egG1Y162基因序列并进行蛋白序列对比分析。方法从细粒棘球蚴原头蚴提取mRNA,mRNA反转录为cDNA。设计特异引物,以cDNA为模板扩增egG1Y162基因,构建PUCm-T/egG1Y162重组质粒,经PCR、酶切及测序鉴定后进行序列分析。结果egG1Y162 cDNA为459bp,编码153个氨基酸;同源性比较表明,egG1Y162 cDNA与emY162同源性为95%,与eg95的同源性为30.61%。结论成功克隆egG1Y162抗原基因,egG1Y162蛋白质氨基酸序列与emY162有很高的相似性,但同其他抗原蛋白质序列存在明显差异,所以egG1Y162是一种新的抗原基因。     

The lack of anti-idiotypic antibodies, not the presence of the corresponding autoantibodies to glutamate decarboxylase, defines type 1 diabetes

Autoantibodies to glutamate decarboxylase 65 (GAD65Ab) are commonly believed to be a major characteristic for type 1 diabetes (T1D). We investigated the presence of GAD65Ab in healthy individuals (n = 238) and first-degree relatives (FDRs) of T1D patients (n = 27) who tested negative for GAD65Ab in conventional RIAs. Sera were applied to affinity columns coated with GAD65-specific mAbs to absorb anti-idiotypic antibodies (anti-Ids). The absorbed sera were analyzed for binding to GAD65 by RIAs. Both healthy individuals and FDRs present GAD65Ab that are inhibited by anti-Id, masking them in conventional detection methods. The presence of GAD65Ab-specific anti-Ids was confirmed by competitive ELISA. Remarkably, T1D patients (n = 54) and Stiff Person Syndrome patients (n = 8) show a specific lack of anti-Ids to disease-associated GAD65Ab epitopes. Purified anti-Ids from healthy individuals and FDRs inhibited the binding of GAD65Ab from T1D patients to GAD65. We conclude that masked GAD65Ab are present in the healthy population and that a lack of particular anti-Ids, rather than GAD65Ab per se, is a characteristic of T1D. The lack of these inhibitory antibodies may contribute to T cell activation by GAD65Ab.     

阴道毛滴虫Rac1蛋白的cDNA克隆和序列分析

目的　获得阴道毛滴虫Rac1蛋白的cDNA克隆,研究其在细胞周期中的调解作用。　方法　提取阴道毛滴虫总RNA,构建cDNA表达文库,随机分离cDNA克隆并测序。用在线生物分析软件NCBIBLAST、ClustalW以及Treeview等程序进行序列分析。　结果　获得一株有714bp的cDNA克隆。序列分析表明,该克隆开放阅读框具600bp,推测肽链具200个氨基酸。该肽链与Rho家族中Rac1鸟苷三磷酸(GTP)酶同源性最高(>60 %),并具多种RhoGTP酶的保守基序,如GTP结合部位、GTP酶激活蛋白作用基序、GTP分离抑制因子作用基序、鸟嘌呤核苷酸交换因子作用基序等。进化树分析显示该克隆属于Rac亚家族GTP酶,与原虫Rac1蛋白最接近。　结论　该克隆属RhoGTP酶的Rac亚家族,很可能是阴道毛滴虫的Rac1蛋白。     

Epitope-restricted 65-kilodalton glutamic acid decarboxylase autoantibodies among new-onset Sardinian type 2 diabetes patients define phenotypes of autoimmune diabetes

The 65-kDa glutamic acid decarboxylase (GAD65) autoantibodies (GAD65Abs), commonly found in type 1 diabetes mellitus (T1DM) patients, are also found at lower frequencies in type 2 diabetes mellitus (T2DM) patients. GAD65Abs in T1DM patients are epitope specific, in contrast to those found in other GAD65Ab-positive individuals, including T2DM patients. Our aim was to assess whether epitope-specific GAD65Abs, or the additional presence of islet antigen 2 (IA-2) autoantibodies, better define T1DM phenotypes among T2DM patients. GAD65 and IA-2 autoantibodies were analyzed in 1436 Sardinian subjects classified with T2DM and in 384 nondiabetic patient controls. Autoantibody binding specificity to the N-terminal, middle (M), and C-terminal (C) portions of the GAD65 molecule was evaluated. Among the T2DM patients, 5.1% had GAD65 (P < 0.001) and 2.4% had IA-2 autoantibodies, compared with 1.3 and 1.6%, respectively, among the controls. GAD65Ab-positive T2DM patients with M+C (epitope-specific) reactivity were found to have the lowest body mass index (P < 0.001), followed by GAD65Ab/IA-2Ab-positive patients (P < 0.01), and non-M+C-reactive (non-epitope-specific) patients (P < 0.02). In GAD65Ab-positive T2DM patients, c-peptide levels were lower in M+C-reactive compared with non-M+C-reactive patients. Sardinian T2DM patients with M+C-predominant GAD65Ab reactivity have clinical features more similar to those of T1DM patients. Thus, GAD65Ab epitope analysis may help to define T1DM phenotypes among newly diagnosed GAD65Ab-positive patients classified with T2DM.     

Cloning, sequence, and expression of bovine interleukin 2.

Interleukin 2 (IL-2) cDNA clones have been isolated from both human and murine sources. We report here the isolation of a cDNA clone encoding bovine IL-2. This was accomplished by screening a cDNA library constructed from lectin-stimulated bovine lymph node cells, using a human IL-2 probe. Bovine IL-2 is composed of 155 amino acids and has a predicted molecular weight of 19,555. Alignment of the amino acid sequence with human IL-2 indicates that mature bovine IL-2 is composed of 135 amino acids and has a predicted molecular weight of 15,452. It has an amino acid homology of 65% with human IL-2 and 50% with murine IL-2. Bovine IL-2 is unique among IL-2 homologs in that it has a single N-linked glycosylation site. Biologically active bovine IL-2 was synthesized in an Escherichia coli expression system.     

Early epitope- and isotype-specific humoral immune responses to GAD65 in young children with genetic susceptibility to type 1 diabetes

OBJECTIVE: The pattern of the humoral immunity to disease-associated autoantigens may reflect the severity of the autoimmune disease process. The purpose of this study was to delineate the maturation of the humoral immunity to one of the main autoantigens in type 1 diabetes (T1D), glutamic acid decarboxylase (GAD65). DESIGN AND METHODS: Serum samples were obtained for the detection of epitope- and isotype-specific antibodies sequentially with short intervals from 36 young children with HLA-conferred genetic susceptibility to T1D starting from the first appearance of GAD65Ab. During prospective observation, ten children developed T1D. Antibodies were analyzed using biotinylated anti-human immunoglobulin (Ig) antibodies and chimeric GAD molecules in radio-binding assays. RESULTS: The immune response to GAD65 started as reactivity to the middle region and spread rapidly to the C-terminal region. IgG1 antibodies dominated among the isotypes from the first appearance of GAD65Ab, while other IgG subclasses were observed to a lesser extent. IgG4 antibodies emerged clearly as the last IgG subclass. A broad initial response comprising three to four IgG subclasses and the lack of an emerging IgG4 response during follow-up was associated with increased risk for progression to clinical diabetes (P<0.05). CONCLUSIONS: The humoral response to GAD65 epitope clusters is relatively uniform in young children, whereas there is conspicuous individual variation in IgG subclass responses except for IgG1. A narrow initial IgG subclass response to GAD65 and the emergence of IgG4 antibodies were characteristic of those who remained non-diabetic over the first few years of GAD65 autoimmunity.     

乙型肝炎病毒X蛋白反式激活基因克隆化的研究

目的 应用抑制性消减杂交（SSH）技术构建乙型肝炎病毒X蛋白（HBX）反式激活基因差异表达的cDNA消减文库，克隆HBX反式激活相关基因。方法 以HBX表达质粒pcDNA3.1(-)-X转染HepG2细胞，以空载体pcDNA3.1(-)转染的HepG2细胞为对照。制备转染后的细胞裂解液，提取mRNA并逆转录为cDNA，经RsaⅠ酶切后，将实验组cDNA分成两组。分别与两组不同的接头衔接，再对照组cDNA进行两次消减杂交及两次抑制聚合酶链反应（PCR），将产物与T/A载体连接，构建cDNA消减文库，并转染大肠杆菌进行文库扩增，随机挑选克隆PCR扩增后进行测序及同源性分析。结果 成功构建人HBX反式激活基因差异表达的cDNA消减文库；文库扩增后得到85个白色克隆，进行菌落PCR分析。均得以200-1000bp插入片段，挑取含有插入片段的65个克隆进行测序，并通过生物信息学分析获得19种已知基因序列。和15个未知基因。结论 应用SSH技术成功构建了HBX反式激活基因差异表达的cDNA消减文库，该文库的建立为进一步阐明HBX反式调节的靶基因及致肝细胞癌发生的分子生物学机制提供理论依据。