本文選題：小電導(dǎo)鈣激活鉀通道　切入點：重疊PCR　出處：《瀘州醫(yī)學(xué)院學(xué)報》2016年06期

【摘要】：目的:采用Overlapping PCR(重疊PCR)法進(jìn)行人源SK2通道Flag和GFP融合表達(dá)質(zhì)粒的構(gòu)建,為下一步胞外運用Flag抗體進(jìn)行SK2通道的轉(zhuǎn)運調(diào)控研究奠定基礎(chǔ)。方法:在既往克隆的人SK2通道基因的表達(dá)質(zhì)粒p IRES-SK2的基礎(chǔ)上,采用重疊PCR法構(gòu)建Flag和GFP雙標(biāo)簽標(biāo)記的表達(dá)質(zhì)粒pEGFP-N3-Flag-SK2(簡寫為Flag-SK2-GFP)。結(jié)果:構(gòu)建的表達(dá)質(zhì)粒Flag標(biāo)簽插入SK2通道的S1-S2胞外環(huán)區(qū)域,GFP標(biāo)簽連接SK2通道胞內(nèi)的C末端,通過酶切、測序等證實質(zhì)粒構(gòu)建成功。結(jié)論:成功構(gòu)建人心房肌SK2通道基因表達(dá)質(zhì)粒Flag-SK2-GFP,為下一步研究SK2通道轉(zhuǎn)運過程及調(diào)控機(jī)制奠定了基礎(chǔ)。
[Abstract]:Aim: to construct the fusion expression plasmid of human SK2 channel Flag and GFP by Overlapping PCR, and to lay a foundation for the further study on the regulation of SK2 channel transport by extracellular Flag antibody.Methods: based on the previously cloned expression plasmid p IRES-SK2 of human SK2 channel gene, the expression plasmid pEGFP-N3-Flag-SK2 (Flag-SK2-GFPN) was constructed by overlapping PCR method.Results: the constructed expression plasmid Flag tag was inserted into the S1-S2 outer loop region of the SK2 channel and connected to the C-terminal of the SK2 channel. The plasmid was successfully constructed by restriction endonuclease digestion and sequencing.Conclusion: the gene expression plasmid Flag-SK2-GFPin of human atrial muscle SK2 channel was successfully constructed, which laid a foundation for the further study of SK2 channel transport process and regulation mechanism.
【作者單位】： 西南醫(yī)科大學(xué)心血管醫(yī)學(xué)研究所醫(yī)學(xué)電生理學(xué)教育部重點實驗室四川省心血管疾病防治協(xié)同創(chuàng)新中心;
【基金】：國家自然科學(xué)基金資助項目(NO:31300948;81670310) 四川省科技廳支撐計劃(2011FZ0106) 瀘州市-四川醫(yī)科大學(xué)聯(lián)合資助項目(2015LZCYD-S03)
【分類號】：R91

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