【摘要】：[目的]構(gòu)建靶向干擾內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志性因子CHOP的shRNA真核表達(dá)載體,并檢測其對CHOP的干擾效率。[方法]查詢Gen Bank數(shù)據(jù)庫,獲取人源CHOP基因mRNA的序列,按照小干擾RNA(siRNA)靶序列的設(shè)計原則,設(shè)計并構(gòu)建靶向CHOP基因mRNA的4個特異性shRNA真核表達(dá)載體(shRNA-1、shRNA-2、shRNA-3、shRNA-4)和1個無同源性的陰性對照載體(shRNA-NC),經(jīng)PCR和測序鑒定確認(rèn)shRNA載體構(gòu)建成功后,脂質(zhì)體轉(zhuǎn)染人正常肝細(xì)胞系(L-02),Western Blot法檢測CHOP蛋白的表達(dá),篩選出干擾效果最好的表達(dá)載體。[結(jié)果]PCR和測序結(jié)果顯示,5個shRNA表達(dá)載體均構(gòu)建成功。Western Blot結(jié)果顯示,0.06 g/L衣霉素?fù)p傷24h后,與內(nèi)質(zhì)網(wǎng)應(yīng)激模型組相比,shRNA-1、shRNA-2、shRNA-3、shRNA-4組的CHOP蛋白表達(dá)水平均明顯降低(P0.01),其中shRNA-1和shRNA-4組CHOP干擾效果最明顯。[結(jié)論]構(gòu)建了并成功篩選出靶向干擾CHOP基因的真核表達(dá)載體,為深入研究CHOP介導(dǎo)內(nèi)質(zhì)網(wǎng)應(yīng)激所致細(xì)胞凋亡的信號通路奠定了實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:[objective] to construct shRNA eukaryotic expression vector targeting endoplasmic reticulum stress marker CHOP and detect its interference efficiency to CHOP. [methods] the Gen Bank database was queried to obtain the sequence of human CHOP gene mRNA. According to the design principle of small interference RNA (siRNA) target sequence, four specific shRNA eukaryotic expression vectors (shRNA-1,shRNA-2,shRNA-3,shRNA-4) and one negative control vector (shRNA-NC) targeting CHOP gene mRNA were designed and constructed. The shRNA vector was successfully constructed by PCR and sequencing. The expression of CHOP protein was detected by Liposome transfection into human normal liver cell line (L 鈮，

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