本文選題：shRNA + SP-B��； 參考：《第二軍醫(yī)大學》2012年碩士論文

【摘要】：目的：構建并鑒定針對小鼠SP-B（surfactant protein B，SP-B）基因的序列特異性短發(fā)夾樣RNA（Short hairpin RNA，shRNA）質粒并從中篩選出有效抑制序列。 方法：根據sp-b基因序列及shRNA設計原則，，設計合成4段編碼短發(fā)夾RNA的寡核苷酸序列：shRNA1，shRNA2，shRNA3，shRNA4，以及設計一條亂序非編碼片段shNC做為陰性對照，將其定向克隆到增強綠色熒光蛋白的真核表達載體PGPU6/GFP/Neo中，重組構建shRNA干擾質粒，并對重組質粒進行酶切分析和DNA序列測定。采用脂質體介導的轉染方法將重組質粒轉入MLE-12小鼠肺上皮細胞，熒光顯微鏡觀察并測定轉染效率，實時熒光定量PCR方法檢測轉染后SP-B mRNA的表達水平。 結果：重組質粒表達載體PGPU6/GFP/Neo-SP-B-shRNA經酶切、測序鑒定，顯示設計合成的shRNA編碼序列被成功插入質粒中，測序結果證實插入片段大小和插入方向正確。通過脂質體介導將shRNA1，shRNA2，shRNA3，shRNA4表達載體分別轉染入MLE-12小鼠肺上皮細胞，轉染MLE-12細胞72小時后熒光顯微鏡觀察到細胞內綠色熒光蛋白的表達。實時熒光定量PCR結果顯示：空白對照組sp-b mRNA表達水平與shNC組相比結果無顯著性差異；shRNA干擾組sp-b mRNA表達水平與shNC組相比：其中shRNA1組（t=0.3651,p=0.7335）和shRNA2組（t=0.6082,p=0.5759）與shNC組相比結果無顯著性差異，shRNA3組（t=5.4772,p=0.0054）和shRNA4組（t=3.0408,p=0.0384）與shNC組相比結果有顯著性差異，shRNA3的干擾效果更明顯。 結論：針對小鼠sp-b基因的重組質粒PGPU6/GFP/Neo-SP-B-shRNA1、shRNA2，shRNA3，shRNA4被成功構建，轉染MLE-12細胞后能夠表達綠色熒光蛋白，shRNA3、shRNA4可特異性下調sp-b mRNA表達，其中以shRNA3的靶向抑制作用最強，本實驗構建的重組質粒能有效沉默sp-b基因的表達，從而為研究SP-B shRNA對小鼠肺上皮細胞的影響及研究sp-b基因功能奠定基礎。
[Abstract]:Objective: to construct and identify short hairpin RNA (short hairpin RNA) plasmids targeting mouse SP-B (surfactant protein BmSP-B gene, and to screen out the effective inhibitory sequence from these plasmids. Methods: according to the sp-b gene sequence and shRNA design principle, four oligonucleotide sequences encoding short hairpin RNA were designed and synthesized. The recombinant plasmid was cloned into the eukaryotic expression vector PGPU 6 / GFP / Neo. The shRNA interference plasmid was constructed. The recombinant plasmid was digested and sequenced. The recombinant plasmid was transfected into MLE-12 mouse lung epithelial cells by liposome-mediated transfection. The transfection efficiency was observed by fluorescence microscope and the expression level of SP-B mRNA was detected by real-time fluorescence quantitative PCR. Results: the recombinant plasmid expression vector PGPU6 / GFP-Neo-SP-B-shRNA was digested and sequenced. The result showed that the designed and synthesized shRNA coding sequence was successfully inserted into the plasmid. The result of sequencing confirmed that the inserted fragment size and insertion direction were correct. The expression vector of shRNA1 / shRNA2hRNA3 / shRNA4 was transfected into MLE-12 mouse lung epithelial cells by liposome-mediated transfection. The expression of green fluorescent protein (GFP) in MLE-12 cells was observed by fluorescence microscope 72 hours after transfection. The results of real-time fluorescence quantitative PCR showed that there was no significant difference in the expression level of sp-b between the blank control group and the shNC group. There was no significant difference in the expression of sp-b between shRNA1 and shRNA2 groups compared with shNC group. There was no significant difference between shRNA1 group (tnr 0.3651) and shRNA2 group (tr 0.6082 p0. 5759) and shRNA3 group (t 5. 4772 p0. 0054) and shRNA4 group (tna 3. 0408 p0. 0384). The interference effect of shRNA3 RNAs was more obvious than that of shNC group. Conclusion: the recombinant plasmid PGPU6 / GFP-Neo-SP-B-shRNA1 shRNA2shRNA3hRNA4 was constructed successfully. The recombinant plasmid PGPU6 / Neo-SP-B-shRNA1 shRNA2shRNA3hRNA4 can specifically down-regulate the expression of sp-b mRNA after transfection into MLE-12 cells, among which shRNA3 has the strongest inhibitory effect. The recombinant plasmid can effectively silence the expression of sp-b gene, which lays a foundation for the study of the effect of SP-B shRNA on mouse lung epithelial cells and the function of sp-b gene.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R346

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