大鼠δ阿片受体RNA干扰质粒构建和作用分析
发布时间：2018-10-09 16:03
【摘要】：目的:设计、构建并筛选靶向大鼠DELTA阿片受体(delta opioid receptor, DOR)的shRNA表达载体。 方法:根据大鼠DOR mRNA序列(序列号:NM_012617)设计并体外合成shRNA寡核苷酸片段,退火形成双链,克隆到线性化质粒pGenesil-1,然后进行酶切和测序鉴定。随后用脂质体2000将重组质粒转染大鼠肾上腺嗜铬细胞瘤细胞(PC12细胞),48小时后测定转染效率,并分别用RT-PCR和Western Blot检测重组质粒以及阴性对照和空白对照组mRNA和蛋白表达水平。 结果:酶切证明DOR-shRNA已经插入到质粒载体pGenesil-1里,测序结果证明均为插入正确的克隆质粒,而且质量均符合设计标准。重组质粒转染PC12细胞效率约60%,转染后48小时RT-PCR和WB结果显示,DOR3-shRNA组mRNA和蛋白表达水平明显低于空白对照和阴性对照组。 结论:成功构建了3条靶向大鼠DOR的RNA干扰质粒,并且筛选出DOR3干扰质粒对大鼠DOR基因的抑制作用较强。
[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.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
本文编号：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.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346
【参考文献】
相关期刊论文 前1条
1 张晓燕;张惠斌;黄文龙;;香草酸受体拮抗剂的研究进展[J];中国药师;2006年08期
，本文编号：2259930
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