本文關(guān)鍵詞：Jak2基因兩個新的截短型轉(zhuǎn)錄本的鑒定　出處：《山西醫(yī)科大學》2015年碩士論文　論文類型：學位論文

  更多相關(guān)文章： Jak2基因 選擇性剪接 轉(zhuǎn)錄本

【摘要】：目的:Jak2激酶(Janus kinase)屬于非受體酪氨酸激酶,通過各種受體(如:紅細胞生成素受體)介導的細胞信號轉(zhuǎn)導途徑,在細胞增殖中發(fā)揮著重要的作用。Jak2基因突變?nèi)?Jak2 V617F,Jak2 exon12與骨髓增殖性腫瘤(myeloproliferative neoplasms,MPN)的發(fā)生密切相關(guān)。為了篩查是否還有其它突變位點,我們選取部分MPN確診病例(Jak2 V617F,Jak2 exon12突變均為陰性)進行Jak2基因c DNA全長測序,結(jié)果發(fā)現(xiàn)有新的轉(zhuǎn)錄本存在。本研究旨在證實新轉(zhuǎn)錄本的真實存在,鑒定其RNA結(jié)構(gòu),預測其蛋白結(jié)構(gòu),同時分析其在MPN、AL、CML、健康人及其他物種中的表達情況。方法:1.兩個新轉(zhuǎn)錄本的發(fā)現(xiàn)與鑒定用隨機引物進行逆轉(zhuǎn)錄,為了消除非特異擴增,針對Jak2b和Jak2c的序列在Jak2基因不同位置設計兩對引物F1/R1和F2/R2,通過聚合酶鏈式反應(PCR)聯(lián)合測序鑒定兩個轉(zhuǎn)錄本存在;用oligo(d T)引物鑒定這兩個轉(zhuǎn)錄本是否是帶有Ploy A尾的成熟m RNA。2.兩個新轉(zhuǎn)錄本全長測序分析,對目的RNA進行逆轉(zhuǎn)錄聚合酶鏈反應(RT-PCR),擴增Jak2基因CDS序列,測序鑒定。3.用RT-PCR鑒定兩個新轉(zhuǎn)錄本在不同物種中的表達。結(jié)果:1.用隨機引物和引物F1/R1和F2/R2進行RT-PCR反應,聯(lián)合電泳并測序分析,結(jié)果顯示兩種新轉(zhuǎn)錄本Jak2b和Jak2c是真實存在的;用oligo(d T)引物和引物F1/R1和F2/R2進行RT-PCR反應,同樣聯(lián)合電泳并測序分析,結(jié)果顯示兩種新轉(zhuǎn)錄本Jak2b和Jak2c是成熟的m RNA;在正常人中也檢測到這兩個新轉(zhuǎn)錄本,由此證明轉(zhuǎn)錄本Jak2b和Jak2c是生理性的成熟m RNA。2.轉(zhuǎn)錄本Jak2b是外顯子10的3’端缺失了77個核苷酸,預測形成了421個氨基酸的截短型蛋白。另一個轉(zhuǎn)錄本Jak2c是外顯子10全部缺失,預測形成了415個氨基酸的截短型蛋白。3.野生型Jak2轉(zhuǎn)錄本在哺乳動物中均有表達,在低等生物中不表達,而新轉(zhuǎn)錄本Jak2b和Jak2c在MPN、AL、健康人和新西蘭大白兔中表達,在SD大鼠、小鼠和低等生物中不表達。結(jié)論:1.Jak2基因除了野生型轉(zhuǎn)錄本Jak2a,還存在轉(zhuǎn)錄本Jak2b和Jak2c。轉(zhuǎn)錄本Jak2b是位于Jak2基因第10外顯子3’端77bp的缺失所致,轉(zhuǎn)錄本Jak2c是Jak2基因第10外顯子的缺失所致。2.兩個新轉(zhuǎn)錄本Jak2b和Jak2c是生理性的帶有Ploy(A)尾的成熟m RNA;在不同物種中的表達是有差異的。
[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.

【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R733.3;R730.43

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