【摘要】：目的基因重組構(gòu)建具有穿膜活性的核轉(zhuǎn)錄因子Sox2真核表達(dá)系統(tǒng),將其轉(zhuǎn)染至小鼠角膜基質(zhì)細(xì)胞,檢測(cè)其在小鼠角膜基質(zhì)細(xì)胞中的表達(dá)。方法利用重疊延伸PCR法重組目的基因Sox2-PTD構(gòu)建質(zhì)粒,測(cè)序檢測(cè)重組DNA序列是否克隆正確。確定正確序列后將其轉(zhuǎn)染至小鼠角膜基質(zhì)細(xì)胞。倒置顯微鏡下觀察細(xì)胞生長(zhǎng)情況,轉(zhuǎn)染后約10d免疫熒光化學(xué)鑒定其表達(dá)。結(jié)果成功構(gòu)建質(zhì)粒PCDNA3.1-Sox2-PTD;4個(gè)轉(zhuǎn)錄因子測(cè)序結(jié)果顯示:Sox2基因測(cè)序結(jié)果與基因模板序列100%一致;成功轉(zhuǎn)染至小鼠角膜基質(zhì)細(xì)胞中;熒光顯微鏡下觀察可見(jiàn),Sox2-PTD在細(xì)胞漿和細(xì)胞核內(nèi)均有表達(dá),主要分布在細(xì)胞核內(nèi)。結(jié)論成功構(gòu)建核轉(zhuǎn)錄因子的真核表達(dá)系統(tǒng),可轉(zhuǎn)染至小鼠角膜基質(zhì)細(xì)胞并引起細(xì)胞形態(tài)和表型的變化,為真核重組外源蛋白誘導(dǎo)角膜基質(zhì)細(xì)胞轉(zhuǎn)化為多能干細(xì)胞奠定基礎(chǔ)。
[Abstract]:Objective to construct the eukaryotic expression system of nuclear transcription factor Sox2 with transmembrane activity and transfer it into mouse corneal stroma cells to detect its expression in mouse corneal stroma cells. Methods the recombinant target gene Sox2-PTD was constructed by overlapping extension PCR and sequenced to detect whether the recombinant DNA sequence was cloned correctly. The correct sequence was determined and transferred into mouse corneal stroma cells. The growth of the cells was observed under inverted microscope, and the expression of the cells was identified by immunofluorescence chemistry about 10 days after transfection. Results the sequencing results of plasmid PCDNA3.1-Sox2-PTD;4 transcription factors showed that the sequencing results of Sox2 gene were 100% consistent with those of gene template, and were successfully transformed into mouse corneal stroma cells. Under fluorescence microscope, Sox2-PTD was expressed in cytoplasm and nucleus, mainly distributed in nucleus. Conclusion the eukaryotic expression system of nuclear factor can be transformed into mouse corneal stroma cells and induce morphological and phenotypic changes, which lays a foundation for the transformation of corneal stroma cells into pluripotent stem cells induced by eukaryotic recombinant exogenous protein.
【作者單位】： 鄭州市第二人民醫(yī)院眼科;暨南大學(xué)再生醫(yī)學(xué)教育部重點(diǎn)實(shí)驗(yàn)室 暨南大學(xué)醫(yī)學(xué)院眼科研究所;
【基金】：國(guó)家自然科學(xué)基金資助(編號(hào):30973244) 中央高�；究蒲袠I(yè)務(wù)費(fèi)專項(xiàng)資金資助(編號(hào)21609407) 鄭州市醫(yī)學(xué)博士創(chuàng)業(yè)基金(編號(hào):121PPTGG495-7)~~
【分類號(hào)】：R341

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本文編號(hào)：2519972