大鼠δ阿片受體RNA干擾質(zhì)粒構(gòu)建和作用分析
發(fā)布時間：2018-10-09 16:03
【摘要】：目的:設(shè)計、構(gòu)建并篩選靶向大鼠DELTA阿片受體(delta opioid receptor, DOR)的shRNA表達(dá)載體。 方法:根據(jù)大鼠DOR mRNA序列(序列號:NM_012617)設(shè)計并體外合成shRNA寡核苷酸片段,退火形成雙鏈,克隆到線性化質(zhì)粒pGenesil-1,然后進(jìn)行酶切和測序鑒定。隨后用脂質(zhì)體2000將重組質(zhì)粒轉(zhuǎn)染大鼠腎上腺嗜鉻細(xì)胞瘤細(xì)胞(PC12細(xì)胞),48小時后測定轉(zhuǎn)染效率,并分別用RT-PCR和Western Blot檢測重組質(zhì)粒以及陰性對照和空白對照組mRNA和蛋白表達(dá)水平。 結(jié)果:酶切證明DOR-shRNA已經(jīng)插入到質(zhì)粒載體pGenesil-1里,測序結(jié)果證明均為插入正確的克隆質(zhì)粒,而且質(zhì)量均符合設(shè)計標(biāo)準(zhǔn)。重組質(zhì)粒轉(zhuǎn)染PC12細(xì)胞效率約60%,轉(zhuǎn)染后48小時RT-PCR和WB結(jié)果顯示,DOR3-shRNA組mRNA和蛋白表達(dá)水平明顯低于空白對照和陰性對照組。 結(jié)論:成功構(gòu)建了3條靶向大鼠DOR的RNA干擾質(zhì)粒,并且篩選出DOR3干擾質(zhì)粒對大鼠DOR基因的抑制作用較強(qiáng)。
[Abstract]:Aim: to design, construct and screen shRNA expression vector targeting rat DELTA opioid receptor (delta opioid receptor, DOR). Methods: shRNA oligonucleotide fragments were designed and synthesized according to rat DOR mRNA sequence (serial number: NM012617), annealed to form double strands, cloned into linearized plasmid pGenesil-1, then digested and sequenced. The transfection efficiency of the recombinant plasmid was measured 48 hours after transfection into rat adrenal pheochromocytoma cells (PC12 cells) with liposome 2000. The expression levels of mRNA and protein in the recombinant plasmid, negative control group and blank control group were detected by RT-PCR and Western Blot, respectively. Results: restriction endonuclease digestion showed that DOR-shRNA had been inserted into the plasmid vector pGenesil-1. The sequencing results showed that all the cloned plasmids were inserted correctly and the quality met the design criteria. The efficiency of transfection of recombinant plasmid into PC12 cells was about 60. The results of RT-PCR and WB showed that the expression level of mRNA and protein in DOR3-shRNA group was significantly lower than that in blank control group and negative control group 48 hours after transfection. Conclusion: three RNA interference plasmids targeting rat DOR were successfully constructed and the inhibitory effect of DOR3 interference plasmids on rat DOR gene was strong.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346
本文編號：2259930
[Abstract]:Aim: to design, construct and screen shRNA expression vector targeting rat DELTA opioid receptor (delta opioid receptor, DOR). Methods: shRNA oligonucleotide fragments were designed and synthesized according to rat DOR mRNA sequence (serial number: NM012617), annealed to form double strands, cloned into linearized plasmid pGenesil-1, then digested and sequenced. The transfection efficiency of the recombinant plasmid was measured 48 hours after transfection into rat adrenal pheochromocytoma cells (PC12 cells) with liposome 2000. The expression levels of mRNA and protein in the recombinant plasmid, negative control group and blank control group were detected by RT-PCR and Western Blot, respectively. Results: restriction endonuclease digestion showed that DOR-shRNA had been inserted into the plasmid vector pGenesil-1. The sequencing results showed that all the cloned plasmids were inserted correctly and the quality met the design criteria. The efficiency of transfection of recombinant plasmid into PC12 cells was about 60. The results of RT-PCR and WB showed that the expression level of mRNA and protein in DOR3-shRNA group was significantly lower than that in blank control group and negative control group 48 hours after transfection. Conclusion: three RNA interference plasmids targeting rat DOR were successfully constructed and the inhibitory effect of DOR3 interference plasmids on rat DOR gene was strong.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 張曉燕;張惠斌;黃文龍;;香草酸受體拮抗劑的研究進(jìn)展[J];中國藥師;2006年08期
，本文編號：2259930
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