本文選題：Spag6L基因 + 精子發(fā)生�。� 參考：《武漢科技大學(xué)》2016年碩士論文

【摘要】：目的:構(gòu)建小鼠Spag6L基因的真核表達(dá)質(zhì)粒,并研究其在哺乳動(dòng)物細(xì)胞內(nèi)的表達(dá)與定位。構(gòu)建上游啟動(dòng)子熒光素酶報(bào)告基因重組質(zhì)粒,并檢測(cè)其轉(zhuǎn)錄活性。方法:以小鼠的睪丸c DNA基因文庫(kù)作為模版,PCR擴(kuò)增Spag6L基因全長(zhǎng)序列,測(cè)序正確后亞克隆至p EGFP-N2和pc DNA3真核表達(dá)載體中并酶切鑒定。將構(gòu)建成功的重組表達(dá)質(zhì)粒轉(zhuǎn)染至COS-7與CHO細(xì)胞中,Western-blot法檢測(cè)COS-7細(xì)胞中Spag6L蛋白表達(dá),激光共聚焦掃描顯微鏡觀察Spag6L蛋白在CHO細(xì)胞內(nèi)的定位。構(gòu)建上游啟動(dòng)子熒光素酶報(bào)告基因重組質(zhì)粒Spag6L/PGL3,用雙熒光素酶法檢測(cè)其轉(zhuǎn)錄活性并與Spag6進(jìn)行比較。結(jié)果:重組質(zhì)粒Spag6L/pEGFP和Spag6L/pc DNA3經(jīng)雙酶切后產(chǎn)生的1.5kb的目的插入片段,經(jīng)測(cè)序證實(shí)與Spag6L基因一致。GFP-Spag6L融合蛋白分子量為82k D,Spag6L蛋白分子質(zhì)量為56k D。Spag6L蛋白在CHO細(xì)胞中主要定位于細(xì)胞質(zhì),以微管和囊泡表達(dá)為主。Spag6L上游啟動(dòng)子轉(zhuǎn)錄調(diào)節(jié)序列熒光素酶活性明顯強(qiáng)于Spag6。結(jié)論:構(gòu)建Spag6L/p EGFP、Spag6L/pc DNA3和Spag6L/PGL3重組質(zhì)粒成功,為進(jìn)一步探究Spag6L基因與Spag6基因的關(guān)系以及Spag6L基因在精子發(fā)生中的作用奠定了基礎(chǔ)。
[Abstract]:Aim: to construct the eukaryotic expression plasmid of mouse Spag6L gene and study its expression and localization in mammalian cells. The recombinant plasmid of luciferase reporter gene of upstream promoter was constructed and its transcriptional activity was detected. Methods: the full-length sequence of Spag6L gene was amplified by PCR using mouse testis c DNA library as template, and subcloned into pEGFP-N2 and pcDNA3 eukaryotic expression vectors and identified by restriction endonuclease digestion. The recombinant expression plasmid was transfected into COS-7 and Cho cells to detect the expression of Spag6L protein by Western-blot. The localization of Spag6L protein in Cho cells was observed by confocal laser scanning microscopy. The upstream promoter luciferase reporter gene recombinant plasmid Spag6L / PGL3 was constructed and its transcriptional activity was detected by double luciferase method and compared with that of Spag6. Results: the recombinant plasmids Spag6L / pEGFP and Spag6L / pcDNA3 were inserted into the 1.5kb fragment. The molecular weight of the recombinant plasmid Spag6L / pEGFP and Spag6L / pcDNA3 was 56kD.Spag6L, which was consistent with that of Spag6L gene. The molecular weight of the fusion protein was 82kD / Spag6L, which was mainly located in the cytoplasm of Cho cells. The luciferase activity of transcriptional regulatory sequence of upstream promoter of microtubule and vesicle was significantly higher than that of Spag6L. Conclusion: the recombinant plasmids Spag6L / p EGFPN Spag6L / pcDNA3 and Spag6L / PGL3 were successfully constructed, which laid a foundation for further study of the relationship between Spag6L gene and Spag6 gene and the role of Spag6L gene in spermatogenesis.
【學(xué)位授予單位】：武漢科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R698.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 徐學(xué)虎;黎淑玲;徐遠(yuǎn)東;吳小兵;伍尚標(biāo);陳戎;;ERG基因3'端非翻譯區(qū)熒光素酶報(bào)告載體的構(gòu)建及其活性[J];廣東醫(yī)學(xué);2015年20期

2 龔瑞龍;呂凸;韓慶榮;張玲;王樂(lè)群;許珊丹;朱長(zhǎng)才;;育齡男性生殖系統(tǒng)疾患與行為因素相關(guān)性分析[J];中華疾病控制雜志;2015年08期

3 張仁;張圣潔;張學(xué)研;李彤;臧文巧;;CLPTM1L與miR-494靶向關(guān)系的雙熒光素酶報(bào)告實(shí)驗(yàn)驗(yàn)證[J];鄭州大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2015年03期

4 蔡成云;吳哲;徐振山;宋禮華;;精子DNA完整性與輔助生殖結(jié)局關(guān)系的Meta分析[J];中華疾病控制雜志;2015年03期

5 郭繼梅;呂玲;宮玉環(huán);;男性不育的病因及治療新進(jìn)展[J];醫(yī)學(xué)綜述;2013年22期

6 曹霞;陳云香;楊華;;基于SCI-E數(shù)據(jù)庫(kù)的轉(zhuǎn)化醫(yī)學(xué)文獻(xiàn)計(jì)量分析[J];現(xiàn)代情報(bào);2013年08期

7 聞浩;魯立;;轉(zhuǎn)化醫(yī)學(xué)對(duì)醫(yī)學(xué)期刊辦刊思路的啟示[J];中國(guó)科技期刊研究;2013年01期

8 董爾丹;胡海;洪微;;淺析轉(zhuǎn)化醫(yī)學(xué)與醫(yī)學(xué)實(shí)踐[J];科學(xué)通報(bào);2013年01期

9 張菁;顏磊;于鵬;李明江;趙興波;;SPAG9在子宮腺肌病中的表達(dá)及臨床意義[J];中國(guó)婦產(chǎn)科臨床雜志;2012年05期

10 魏莉;顏磊;張輝;李明江;趙興波;;精子相關(guān)抗原9在子宮平滑肌肉瘤中的表達(dá)及臨床意義[J];現(xiàn)代婦產(chǎn)科進(jìn)展;2012年05期

，

本文編號(hào)：2109934