Jak2基因两个新的截短型转录本的鉴定

发布时间：2018-01-09 01:07

  本文关键词：Jak2基因两个新的截短型转录本的鉴定　出处：《山西医科大学》2015年硕士论文　论文类型：学位论文

  更多相关文章： Jak2基因 选择性剪接 转录本

【摘要】：目的:Jak2激酶(Janus kinase)属于非受体酪氨酸激酶,通过各种受体(如:红细胞生成素受体)介导的细胞信号转导途径,在细胞增殖中发挥着重要的作用。Jak2基因突变如:Jak2 V617F,Jak2 exon12与骨髓增殖性肿瘤(myeloproliferative neoplasms,MPN)的发生密切相关。为了筛查是否还有其它突变位点,我们选取部分MPN确诊病例(Jak2 V617F,Jak2 exon12突变均为阴性)进行Jak2基因c DNA全长测序,结果发现有新的转录本存在。本研究旨在证实新转录本的真实存在,鉴定其RNA结构,预测其蛋白结构,同时分析其在MPN、AL、CML、健康人及其他物种中的表达情况。方法:1.两个新转录本的发现与鉴定用随机引物进行逆转录,为了消除非特异扩增,针对Jak2b和Jak2c的序列在Jak2基因不同位置设计两对引物F1/R1和F2/R2,通过聚合酶链式反应(PCR)联合测序鉴定两个转录本存在;用oligo(d T)引物鉴定这两个转录本是否是带有Ploy A尾的成熟m RNA。2.两个新转录本全长测序分析,对目的RNA进行逆转录聚合酶链反应(RT-PCR),扩增Jak2基因CDS序列,测序鉴定。3.用RT-PCR鉴定两个新转录本在不同物种中的表达。结果:1.用随机引物和引物F1/R1和F2/R2进行RT-PCR反应,联合电泳并测序分析,结果显示两种新转录本Jak2b和Jak2c是真实存在的;用oligo(d T)引物和引物F1/R1和F2/R2进行RT-PCR反应,同样联合电泳并测序分析,结果显示两种新转录本Jak2b和Jak2c是成熟的m RNA;在正常人中也检测到这两个新转录本,由此证明转录本Jak2b和Jak2c是生理性的成熟m RNA。2.转录本Jak2b是外显子10的3’端缺失了77个核苷酸,预测形成了421个氨基酸的截短型蛋白。另一个转录本Jak2c是外显子10全部缺失,预测形成了415个氨基酸的截短型蛋白。3.野生型Jak2转录本在哺乳动物中均有表达,在低等生物中不表达,而新转录本Jak2b和Jak2c在MPN、AL、健康人和新西兰大白兔中表达,在SD大鼠、小鼠和低等生物中不表达。结论:1.Jak2基因除了野生型转录本Jak2a,还存在转录本Jak2b和Jak2c。转录本Jak2b是位于Jak2基因第10外显子3’端77bp的缺失所致,转录本Jak2c是Jak2基因第10外显子的缺失所致。2.两个新转录本Jak2b和Jak2c是生理性的带有Ploy(A)尾的成熟m RNA;在不同物种中的表达是有差异的。
[Abstract]:Objective: Jak2 kinase (Janus kinase) belongs to the non receptor tyrosine kinase, through various receptors (e.g., erythropoietin receptor) cell mediated signal transduction in cell proliferation, plays an important role of.Jak2 gene mutations such as Jak2 V617F, Jak2 exon12 and bone marrow proliferation of tumor (myeloproliferative neoplasms, MPN) is closely related to the occurrence. In order to screen whether there are other mutations, we selected some of the MPN cases (Jak2 V617F, Jak2 exon12 mutations were negative for Jak2 C DNA) gene sequence, and found the new transcripts. The purpose of this study is to confirm the existence of new transcription, identification of the RNA structure. The prediction of protein structure, at the same time in the analysis of MPN, AL, CML, expression of healthy people and other species. Methods: the discovery and identification of 1. new two random primers were used for reverse transcription, in order to eliminate Non specific amplification, the sequence of Jak2b and Jak2c designed two pairs of primers F1/R1 and F2/R2 in different Jak2 gene location by polymerase chain reaction (PCR) combined with sequencing two transcripts; oligo (D T) were identified that two transcripts are analyzed with Ploy A tail mature m RNA.2. two a new full-length transcript sequencing, reverse transcription polymerase chain reaction of RNA (RT-PCR), Jak2 gene was amplified by CDS sequence,.3. RT-PCR sequencing identified two novel transcripts in different species expression. Results: 1. using random primers and primers F1/R1 and F2/R2 were combined with RT-PCR reaction, electrophoresis and sequencing analysis. The results showed that two kinds of new transcripts of Jak2b and Jak2c are real; oligo (D T) primers and primers F1/R1 and F2/R2 were the same with RT-PCR reaction, electrophoresis and sequencing analysis, the results showed that two kinds of new transcription of the Jak2b and Jak2c are M RNA cooked; in normal people was also detected in the two new transcripts, thus proving that the transcripts of Jak2b and Jak2c is m RNA.2. physiological mature transcription the Jak2b exon 10 of the 3 'end of the deletion of 77 nucleotides, predict the formation of a truncated protein of 421 amino acids. The other one this is a transcription of Jak2c exon 10 deletion of all, predict the formation of a truncated protein of.3. wild-type Jak2 transcription of 415 amino acids in mammals have the expression, no expression in lower organisms, and new transcripts of Jak2b and Jak2c in MPN, AL, table of healthy people and the New Zealand rabbits. In SD rats, expression of mice and lower organisms. Conclusion: 1.Jak2 gene in wild type transcripts of Jak2a, there are the transcripts of Jak2b and Jak2c. transcripts Jak2b is located in the tenth exon of Jak2 gene 3' end of 77bp due to the lack of the Jak2c is missing, the transcription of Jak2 gene exon tenth. The two new transcriptional versions of.2., Jak2b and Jak2c, are physiological mature m RNA with Ploy (A) tail; the expression in different species is different.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.3;R730.43

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