Thyroxine-binding globulin, Molecular biology of the gene and its abnormal expressions

1.甲状腺素结合球蛋白（TBG）cDNA的克隆和序列测定：用抗TBG抗体筛选在gt 11噬菌体中构建的人肝cDNA文库。从阳性克隆中分离cDNA插入片段，并将其重新克隆到质粒pSP 65中。在大规模制备插入片段后，通过双脱氧链终止法测定序列。结果表明，TBG由395个氨基酸组成，含有5个天冬酰胺残基，可以接受糖残基.正常肝和肝癌细胞系HepG 2中TBG mRNA的分析：对从正常人肝和肝癌细胞系提取的RNA进行北方印迹分析。发现两种大小不同的TBG mRNA（2.0和1.8Kb），经区域特异性探针分析，发现这两种mRNA均为交替多聚腺苷酸化产生.基因组TBG基因的克隆：用EcoRI消化从正常人受试者提取的DNA。将含有TBG基因（10-20 Kb）的片段克隆到大肠杆菌中， ...更多信息 经琼脂糖凝胶电泳纯化后，插入噬菌体EMBL-4的臂中。经体外包装后，用TBG cDNA探针筛选基因组文库。基因组TBG克隆包含所有含有所有编码序列的克隆。通过对克隆的基因组TBG基因的分析，发现TBG包含在5 kb的序列中，由4个xons组成. TBG完全缺乏症家系TBG基因分析：对6个TBG完全缺乏症家系进行TBG基因限制性片段长度多态性分析。结果表明，这些家系中TBG缺失并非由TBG基因的缺失、插入或突变所致. TBG过量家系TBG基因分析：对1个TBG过量家系进行限制性内切酶片段长度多态性和基因剂量分析。发现TBG基因扩增可能是导致患者TBG过量的原因. TBG异常（TBG-Gary）的TBG基因分析：TBG-Gary以极低的甲状腺素结合率和IEF阳极偏移为特征。从受影响的受试者的基因组TBG基因克隆和测序分析。结果发现TBG基因第一外显子的单点突变导致第96位氨基酸由异亮氨酸变为天冬酰胺。由于该基因的其他部分没有异常，推测该突变是该家族TBG异常的原因。少

1. Cloning and sequencing of a cDNA coding for thyroxine-binding globulin (TBG): Human liver cDNA library constructed in bacteriophage gtll was screened with anti-TBG antibody. The cDNA insert form positive clone was isolated and recloned in plasmid pSP65. after large scale preparation of the insert, the sequence was determined by dideoxy chain termination method. It was revealed that TBG consist of 395 amino acid and contains 5 asparagine residue which can accept carbohydrate residues.2. Analysis of TBG mRNA in normal liver and hepatoma cell line HepG2: RNAs extracted form normal human liver and hepatoma cell line was subjected to Northern blot analysis. Two TBG mRNA species of different size (2.0 and 1.8 Kb) were found. by the analysis with region specific probe, it was found that the two mRNA was produced by alternative polyadenylation.3. Cloning of Genomic TBG gene: DNA extracted from normal human subject was digested with EcoRI. The fragments containing TBG gene (10-20 Kb) were pu … More rified by agarose gel electrophorsis and inserted into the arms of bacteriophage EMBL-4. After in vitro packaging, the genomic library was screened with TBG cDNA probe. Genomic TBG clone containing all containing all the coding sequence was cloned. by the analysis of the cloned genomic TBG gene, it was found that TBG was contained in 5 kb sequence and consists of 4 xons.4. Analysis of TBG gene in complete TBG deficient families: Restriction fragment length polymorphism of the TBG gene was wvaluated in 6 families with complete TBG deficiency. It was found that the absence of TBG in these families were not caused by gross deletion, insertion or mutation in the TBG gene.5. Analysis of TBG gene in TBG excess family: Restriction fragment length polymorphism and gene dosage was evaluated in a family with TBG excess. it was found that TBG excess in the patients might be caused by the amplification of TBG gene.6. Analysis of TBG gene in abnormal TBG (TBG-Gary): TBG-Gary was characterized by extrem ely low thyroxine-binding and anodal shift on IEF. Genomic TBG gene from an affected subject was cloned and analyzed by sequencing. It was found that single point mutation in the first exon of TBG fene resulted in the substitution of amino acid 96 from isoleucine to asparagine. Since there was no abnormality in other part of the gene, it was speculated that this mutation is the cause of abnormal TBG in the family. Less