【摘要】：[目的]構建靶向干擾內質網應激標志性因子CHOP的shRNA真核表達載體,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)靶序列的設計原則,設計并構建靶向CHOP基因mRNA的4個特異性shRNA真核表達載體(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PCR和測序鑒定確認shRNA載體構建成功后,脂質體轉染人正常肝細胞系(L-02),Western Blot法檢測CHOP蛋白的表達,篩選出干擾效果最好的表達載體。[結果]PCR和測序結果顯示,5個shRNA表達載體均構建成功。Western Blot結果顯示,0.06 g/L衣霉素損傷24h后,與內質網應激模型組相比,shRNA-1、shRNA-2、shRNA-3、shRNA-4組的CHOP蛋白表達水平均明顯降低(P0.01),其中shRNA-1和shRNA-4組CHOP干擾效果最明顯。[結論]構建了并成功篩選出靶向干擾CHOP基因的真核表達載體,為深入研究CHOP介導內質網應激所致細胞凋亡的信號通路奠定了實驗基礎。
[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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