RET基因真核表达载体的构建及其体外定点突变

发布时间：2018-04-08 19:38

本文选题：RET　切入点：载体　出处：《郑州大学》2005年硕士论文

【摘要】：RET(Rearranged during transfection)基因是1985年发现的在基因转染过程中可以重排的基因。它具有20个外显子。其中1号外显子约为24kb,2-20号外显子总计31kb。 RET转录产物经过可变剪切后,形成5种RNA分子,经过翻译及翻译后加工,可形成3种同型Ret蛋白。研究表明,大鼠胚胎早期,Ret高度表达于周围、中枢神经系统、排泄系统等。胚胎中期,第四菱脑结节神经嵴细胞、胚肾管细胞、肾上腺嗜铬祖细胞、甲状旁腺祖细胞均可发现RETmRNA高转录活性。 Ret蛋白是受体酪氨酸激酶(receptor turosine kinase,RTK)家族的一个新成员,为典型的TK结构。Ret蛋白是胶质细胞源性神经生长因子(GDNF)的受体,其主要的功能是将GDNF的信息向细胞内传递。当GDNF-GFRas-Ret复合物形成之后,Ret发生磷酸化激活,然后启动多种途径,向细胞内传递信息,从而调节神经嵴细胞的增殖、分化、以及迁移到肠壁。RET的保守性及表达状况是肠神经系统、肾脏输尿管芽形成及肾组织器官发育的早期遗传学事件。研究表明,RET基因的突变、重排及缺失,与多发性内分泌腺瘤、乳突状甲状腺癌及先天性巨结肠密切相关。在RET相关疾病中,多发性内分泌瘤2B(MEN2B)型病情最为严重。RET基因点突变是该病发生的分子事件。95%的患者的突变位点位于第16号外显子上的918位密码子处(cDNA第2753个碱基,由T变为C),即Met(ATG)变为Thr(ACG)。由于该处是酶的催化中心,突变直接导致Ret蛋白胞内部分构象发生变化。在与没有与相应配体结合时,自发激活,形成二聚体,产生磷酸化。最终激活下游信号分子,向胞内传递信息,诱导细胞增生过度以至癌变。关于MEN2B的研究,国内外基本上局限于通过RT-PCR的方法获得cDNA
[Abstract]:RET(Rearranged during transfectiongene was discovered in 1985 and can be rearranged during gene transfection.It has 20 exons.Exon 1 is about 24 kb / 2-20 exon in total 31 kb.Five RNA molecules were formed from RET transcripts after variable splicing. After translation and post-translational processing, three homotypic Ret proteins were formed.It has been shown that Ret is highly expressed in the peripheral central nervous system and excretory system in early embryonic rat.The high transcriptional activity of RETmRNA was found in midembryonic, fourth rhombohedral nodule neural crest cells, embryonic renal duct cells, adrenal chromaffin progenitor cells and parathyroid progenitor cells.Ret protein is a new member of the receptor tyrosine kinase receptor turosine kinase- rk family. It is a typical TK structure. Ret protein is the receptor of glial cell-derived nerve growth factor (GDNF). Its main function is to transfer the information of GDNF to the cells.After the formation of GDNF-GFRas-Ret complex, the activation of phosphorylation of ret and the initiation of multiple pathways to transmit information to the cells, thereby regulating the proliferation, differentiation, migration to the intestinal wall of the neural crest cells, and the conserved and expressed state of the intestinal nervous system.Early genetic events of renal ureteral bud formation and renal tissue and organ development.Studies have shown that mutations, rearrangements and deletions of the RET gene are closely associated with multiple endocrine adenomas, mastoid thyroid carcinoma and congenital megacolon.Among the RET related diseases, the most serious type of multiple endocrine tumor, 2Bmne2B). RET gene point mutation is the molecular event of the disease. 95% of the patients have mutation locus at the 918 codon on exon 16, which is located at the 2753 base of the codon.From T to C, I. e., Meta ATG, to THR ACGN.Because this site is the catalytic center of the enzyme, mutation directly leads to some conformation changes of Ret protein.When the ligand is not bound to the corresponding ligand, it spontaneously activates, forms dimer and produces phosphorylation.Finally, the downstream signaling molecules are activated to transmit information to the cell and induce hyperproliferation and carcinogenesis.The research on MEN2B is basically limited to obtaining cDNA by means of RT-PCR at home and abroad.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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