本文關(guān)鍵詞：CT45-5在DNA損傷應(yīng)答中作用初探　出處：《福建農(nóng)林大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

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【摘要】：腫瘤-睪丸抗原45-5(Cancer/testis antigen family45, member A5)是Chen等使用大量平行測(cè)序技術(shù)和癌/睪丸限制性mRNA表達(dá)模型鑒定出的CT抗原基因，這種方法發(fā)現(xiàn)了超過20種CT或者CT樣基因，其中包括CT45。CT45位于X染色體著絲粒末端Xq26.3，其中包括6個(gè)幾乎相同的基因拷貝。CT45-5基因全長(zhǎng)為567bp，編碼189個(gè)氨基酸。本課題組先前利用基因芯片技術(shù)進(jìn)行基因差異表達(dá)分析顯示隨著FHIT基因的高表達(dá)，有一系列基因的表達(dá)也出現(xiàn)上調(diào)，其中CT45-5的上調(diào)最為明顯，對(duì)其進(jìn)行了RNA水平和蛋白水平的驗(yàn)證，并且制備了多克隆抗體。 我們前期的研究發(fā)現(xiàn)Fhit-/-細(xì)胞對(duì)DNA損傷誘導(dǎo)劑具有更強(qiáng)的耐受性，為了進(jìn)一步研究CT45-5和Fhit之間的關(guān)系以及CT45家族在DNA損傷應(yīng)答中的作用，本課題首先根據(jù)CT45-5基因序列，通過生物信息學(xué)的方法對(duì)靶向CT45-5基因的siRNA進(jìn)行預(yù)測(cè)，，從中篩選出2對(duì)合理的CT45-5-siRNA，將其克隆到siRNA表達(dá)載體pSilencer2.1-U6Hygro中，篩選得到陽(yáng)性克隆，序列分析得到一條正確的CT45-5-siRNA。用構(gòu)建成功的CT45-5-siRNA重組載體轉(zhuǎn)染人宮頸癌細(xì)胞HeLa及HeLa/Fhit(過表達(dá)Fhit的HeLa細(xì)胞，3-18)，Western印跡檢測(cè)siRNA對(duì)HeLa及HeLa/Fhit內(nèi)源性CT45-5以及外源FHIT基因表達(dá)的干擾效果，結(jié)果顯示該siRNA對(duì)CT45-5基因具有明顯的干擾作用，并且FHIT基因表達(dá)也被抑制，提示CT45-5與Fhit可能存在某種功能上的聯(lián)系。 隨后，我們對(duì)靶向CT45-5基因的siRNA的功能進(jìn)行了研究。通過細(xì)胞周期實(shí)驗(yàn)，證明了在對(duì)細(xì)胞進(jìn)行輻射后，轉(zhuǎn)染靶向CT45-5基因的siRNA的細(xì)胞出現(xiàn)了明顯的G2期延遲現(xiàn)象；通過MTT實(shí)驗(yàn)，我們證明了在DNA損傷誘導(dǎo)劑處理的情況下，用靶向CT45-5基因的siRNA表達(dá)質(zhì)粒與空載體分別轉(zhuǎn)染過表達(dá)Fhit的HeLa細(xì)胞(3-18)，前者對(duì)DNA損傷誘導(dǎo)劑具有更強(qiáng)的耐受性，而轉(zhuǎn)染沒有Fhit表達(dá)的HeLa細(xì)胞則不受DNA損傷誘導(dǎo)劑的影響。實(shí)驗(yàn)結(jié)果提示我們靶向CT45-5基因的siRNA在細(xì)胞中可能通過抑制Fhit的表達(dá)，從而提高細(xì)胞對(duì)DNA損傷誘導(dǎo)劑的耐受性。 本實(shí)驗(yàn)通過對(duì)CT45-5基因的siRNA表達(dá)質(zhì)粒的研究，得到了一條CT45-5-siRNA，為進(jìn)一步研究Fhit及CT45-5在DNA損傷應(yīng)答中的作用機(jī)制奠定了基礎(chǔ)。
[Abstract]:Cancer testis antigen 45-5 (Cancer/testis antigen family45, member A5) CT antigen gene Chen using massively parallel sequencing and expression of cancer / testis restricted mRNA model identified, this method has found more than 20 kinds of CT or CT like genes, including CT45.CT45 X located in the centromeric end of Xq26.3, including 6 almost the same gene copy of.CT45-5 gene was 567bp, encoding 189 amino acids. The expression analysis showed that with the high expression of FHIT gene in our previous use of gene chip technology has a series of genes, gene expression is also up-regulated, the upregulation of CT45-5 was the most obvious, which is verified by RNA and the protein level, and preparation of the polyclonal antibody.
Fhit-/- cell damage tolerance inducing agent is more of DNA found in our previous study, in order to further study the relationship between CT45-5 and Fhit and CT45 in the family in response to DNA damage, firstly, according to the sequence of CT45-5 gene were predicted by Bioinformatics Method of siRNA targeting CT45-5 gene screening. 2 of the reasonable from the CT45-5-siRNA, cloned into siRNA expression vector pSilencer2.1-U6Hygro. The positive clones were screened for sequence analysis, get a correct CT45-5-siRNA. constructed CT45-5-siRNA recombinant vector was transfected into human cervical carcinoma cells (HeLa and HeLa/Fhit Fhit expression in HeLa cells, 3-18, siRNA) Western blot on the interference effect of HeLa and HeLa/Fhit CT45-5 and endogenous exogenous FHIT gene expression, the results show that the siRNA has obvious interference on CT45-5 gene function, and The expression of FHIT gene is also inhibited, suggesting that CT45-5 and Fhit may have some functional relationship.
Subsequently, we targeting siRNA CT45-5 gene function was studied. Through the cell cycle experiments proved that radiation on cells after transfection targeting siRNA gene of CT45-5 cells appeared obvious G2 phase delay phenomenon; through the MTT experiment, we proved that DNA damage inducer treatment case, plasmid and empty vector were transfected into HeLa cells by overexpression of Fhit target expression of siRNA CT45-5 gene (3-18), the former damage tolerance inducing agent has stronger on DNA, but no Fhit expression transfection HeLa cells was not affected by DNA damage inducer. The experimental results suggest that our siRNA targeting CT45-5 the gene may inhibit the expression of Fhit in cells, so as to improve the cell damage tolerance inducing agent of DNA.
In this study, we obtained a CT45-5-siRNA by studying the siRNA expression plasmid of CT45-5 gene, which laid the foundation for further studying the mechanism of Fhit and CT45-5 in DNA damage response.

【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.1

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相關(guān)期刊論文 前1條

1 胡寶成;;脆性組氨酸三聯(lián)體基因研究進(jìn)展[J];生物技術(shù)通訊;2005年06期

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