小鼠SP-B shRNA真核表达载体的构建、鉴定及有效序的筛选

发布时间：2018-06-22 08:25

  本文选题：shRNA + SP-B　； 参考：《第二军医大学》2012年硕士论文

【摘要】：目的：构建并鉴定针对小鼠SP-B（surfactant protein B，SP-B）基因的序列特异性短发夹样RNA（Short hairpin RNA，shRNA）质粒并从中筛选出有效抑制序列。 方法：根据sp-b基因序列及shRNA设计原则，，设计合成4段编码短发夹RNA的寡核苷酸序列：shRNA1，shRNA2，shRNA3，shRNA4，以及设计一条乱序非编码片段shNC做为阴性对照，将其定向克隆到增强绿色荧光蛋白的真核表达载体PGPU6/GFP/Neo中，重组构建shRNA干扰质粒，并对重组质粒进行酶切分析和DNA序列测定。采用脂质体介导的转染方法将重组质粒转入MLE-12小鼠肺上皮细胞，荧光显微镜观察并测定转染效率，实时荧光定量PCR方法检测转染后SP-B mRNA的表达水平。 结果：重组质粒表达载体PGPU6/GFP/Neo-SP-B-shRNA经酶切、测序鉴定，显示设计合成的shRNA编码序列被成功插入质粒中，测序结果证实插入片段大小和插入方向正确。通过脂质体介导将shRNA1，shRNA2，shRNA3，shRNA4表达载体分别转染入MLE-12小鼠肺上皮细胞，转染MLE-12细胞72小时后荧光显微镜观察到细胞内绿色荧光蛋白的表达。实时荧光定量PCR结果显示：空白对照组sp-b mRNA表达水平与shNC组相比结果无显著性差异；shRNA干扰组sp-b mRNA表达水平与shNC组相比：其中shRNA1组（t=0.3651,p=0.7335）和shRNA2组（t=0.6082,p=0.5759）与shNC组相比结果无显著性差异，shRNA3组（t=5.4772,p=0.0054）和shRNA4组（t=3.0408,p=0.0384）与shNC组相比结果有显著性差异，shRNA3的干扰效果更明显。 结论：针对小鼠sp-b基因的重组质粒PGPU6/GFP/Neo-SP-B-shRNA1、shRNA2，shRNA3，shRNA4被成功构建，转染MLE-12细胞后能够表达绿色荧光蛋白，shRNA3、shRNA4可特异性下调sp-b mRNA表达，其中以shRNA3的靶向抑制作用最强，本实验构建的重组质粒能有效沉默sp-b基因的表达，从而为研究SP-B shRNA对小鼠肺上皮细胞的影响及研究sp-b基因功能奠定基础。
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

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