本文關(guān)鍵詞：野生型和突變型ABCB6紅色熒光融合蛋白表達(dá)載體的構(gòu)建　出處：《醫(yī)學(xué)研究生學(xué)報(bào)》2016年09期 　論文類(lèi)型：期刊論文

  更多相關(guān)文章： ABCB 融合蛋白 表達(dá)載體 細(xì)胞定位

【摘要】：目的 ABCB6基因突變與多種疾病相關(guān),尤其是遺傳性泛發(fā)型色素異常癥。為進(jìn)一步研究ABCB6在色素代謝中的作用,構(gòu)建野生型和突變型ABCB6紅色熒光蛋白融合蛋白表達(dá)載體p DsRed-wt-ABCB6和p DsRed-L356P-ABCB6,并觀察ABCB6在人黑素細(xì)胞瘤細(xì)胞系A(chǔ)375中細(xì)胞定位情況。方法 以前期構(gòu)建的p IRES2-Zs Green1-ABCB6表達(dá)載體為基礎(chǔ)構(gòu)建野生型和突變型ABCB6蛋白與紅色熒光蛋白融合表達(dá)載體p DsRed-wt/L356P-ABCB6,將構(gòu)建的質(zhì)粒轉(zhuǎn)染A375細(xì)胞,Western blot檢測(cè)細(xì)胞中ABCB6的表達(dá)水平,激光共聚焦顯微鏡觀察ABCB6在細(xì)胞中的定位。結(jié)果 p DsRed-wt/L356P-ABCB6質(zhì)粒經(jīng)菌落PCR、雙酶切和測(cè)序鑒定正確,轉(zhuǎn)染A375細(xì)胞后,Western blot檢測(cè)細(xì)胞中ABCB6表達(dá)量顯著增高,激光共聚焦顯微鏡觀察到野生型和突變型ABCB6均定位于細(xì)胞質(zhì)中。結(jié)論 p DsRed-wt/L356P-ABCB6融合蛋白表達(dá)載體成功構(gòu)建,觀察了ABCB6在細(xì)胞中的定位,為進(jìn)一步研究ABCB6的功能及其在相關(guān)疾病中致病機(jī)制奠定了良好的基礎(chǔ)。
[Abstract]:Objective to study the role of ABCB6 gene mutation in pigment metabolism, especially in hereditary generalized dystrophy. Wild-type and mutants of ABCB6 red fluorescent protein fusion protein expression vector p DsRed-wt-ABCB6 and p DsRed-L356P-ABCB6 were constructed. The localization of ABCB6 in human melanoma cell line A375 was observed. Construction of Fusion expression Vector of Wild and Mutant ABCB6 protein and Red fluorescent protein based on Green1-ABCB6 expression Vector. DsRed-wt/L356P-ABCB6. The expression of ABCB6 in A375 cells was detected by Western blot. The localization of ABCB6 in cells was observed by laser confocal microscope. Results the p DsRed-wt/L356P-ABCB6 plasmid was confirmed by colony PCR, double enzyme digestion and sequencing. After transfection of A375 cells, the expression of ABCB6 in A375 cells was detected by Western blot. Both wild-type and mutant ABCB6 were observed in cytoplasm by laser confocal microscopy. Conclusion the expression vector of p DsRed-wt/L356P-ABCB6 fusion protein was successfully constructed. The localization of ABCB6 in cells was observed, which laid a good foundation for further study on the function of ABCB6 and its pathogenesis in related diseases.
【作者單位】： 華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬同濟(jì)醫(yī)院皮膚科;華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬同濟(jì)醫(yī)院麻醉科;
【分類(lèi)號(hào)】：R596
【正文快照】： 0引言ATP結(jié)合盒(ATP-binding cassette,ABC)轉(zhuǎn)運(yùn)蛋白是一類(lèi)大型跨膜蛋白,具有多個(gè)結(jié)構(gòu)域,可通過(guò)ATP依賴(lài)的途徑促進(jìn)多種生物化合物,如藥物、膽酸、多肽、類(lèi)固醇、離子和磷脂等物質(zhì)的跨膜轉(zhuǎn)運(yùn)[1]。人類(lèi)基因組可編碼48種ABC轉(zhuǎn)運(yùn)蛋白,根據(jù)蛋白的功能和同源性可分為7個(gè)亞家族(ABCA，

本文編號(hào)：1408634