SEDL基因重组腺病毒的构建及其对hFOB1.19细胞相关基因和蛋白表达的研究

发布时间：2017-12-31 12:16

本文关键词：SEDL基因重组腺病毒的构建及其对hFOB1.19细胞相关基因和蛋白表达的研究　出处：《重庆医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的构建含SEDL基因的重组腺病毒质粒，收获高滴度的重组腺病毒。采用SEDL基因重组腺病毒增强人SV40转染成骨细胞（hFOB1.19）中SEDL mRNA及sedlin蛋白的表达，为探讨SEDL基因及sedlin蛋白在骨骼代谢中的作用奠定基础。方法采用定向克隆的方法将SEDL基因插入穿梭质粒pAdtrace-TO4，酶切及基因测序鉴定后命名为pAdtrace-SEDL；然后采用同源重组方法将pAdtrace-SEDL与腺病毒骨架质粒pAdEasy-l连接，构建重组腺病毒质粒pAd5-SEDL。酶切鉴定pAd5-SEDL后，用PolyJet将其导入HEK293包装细胞，乒乓感染后获得高滴度的重组腺病毒pAd5-SEDL。应用Ad5-SEDL感染hFOB1.19细胞，实验分两组：实验组（hFOB1.19/Ad5-SEDL）、空白对照组（hFOB1.19）。通过MTT法检测hFOB1.19细胞增殖；RT-PCR方法分别检测hFOB1.19细胞内SEDLmRNA表达；Western Blot法检测hFOB1.19细胞内sedlin蛋白表达。结果（1）酶切及基因测序证实重组腺病毒质粒pAd5-SEDL构建成功。(2)荧光显微镜证实重组腺病毒pAd5-SEDL成功导入HEK293包装细胞。（3）收获高滴度重组腺病毒pAd5-SEDL，，病毒滴度约为4.3×l011pfu/ml。（4）两组细胞中均有SEDLmRNA及sedlin蛋白的表达。（5）实验组SEDL mRNA表达量为（0.79±0.12）与空白对照组（0.11±0.04）相比明显增加，差异有统计学意义（p0.01）。（6）实验组sedlin蛋白表达量为（0.93±0.30），与空白对照组（0.12±0.04）相比也明显增加（p0.01）。结论（1）成功构建了携带SEDL基因的重组腺病毒质粒，收获高滴度重组腺病毒pAd5-SEDL，并成功感染hFOB1.19细胞。（2）hFOB1.19细胞表达SEDL mRNA及sedlin蛋白。（3）携带SEDL基因的重组腺病毒Ad5-SEDL可明显增强hFOB1.19细胞SEDL mRNA及sedlin蛋白的表达。
[Abstract]:Purpose The recombinant adenovirus plasmid containing SEDL gene was constructed. High titer recombinant adenovirus was harvested. SEDL gene recombinant adenovirus was used to enhance the transfection of human SV40 into osteoblast hFOB1.19). The expression of SEDL mRNA and sedlin protein. To study the role of SEDL gene and sedlin protein in bone metabolism. Method The SEDL gene was inserted into the shuttle plasmid pAdtrace-TO4 by directional cloning and named as pAdtrace-SEDL after restriction endonuclease digestion and gene sequencing. Then pAdtrace-SEDL was ligated with adenovirus skeleton plasmid pAdEasy-l by homologous recombination. The recombinant adenovirus plasmid pAd5-SEDL. was constructed and digested to identify the pAd5-SEDL. Then the recombinant adenovirus plasmid pAd5-SEDL. was transfected into HEK293 packaging cells by PolyJet. High titer recombinant adenovirus pAd5-SEDLwas obtained after ping-pong infection. HFOB1.19 cells were infected with Ad5-SEDL. The experiment was divided into two groups: hFOB 1.19 / Ad5-SEDLL in the experimental group and hFOB 1.19 in the blank control group. The proliferation of hFOB1.19 cells was detected by MTT assay. The expression of SEDLmRNA in hFOB1.19 cells was detected by RT-PCR method. The expression of sedlin protein in hFOB1.19 cells was detected by Western Blot assay. Results The recombinant adenovirus plasmid pAd5-SEDL was successfully constructed by restriction endonuclease digestion and gene sequencing. Fluorescence microscopy confirmed that the recombinant adenovirus pAd5-SEDL was successfully introduced into HEK293 packaging cell. 3) the recombinant adenovirus pAd5-SEDL was harvested with high titer. The virus titer was about 4.3 脳 10 11 pfuml 路ml. 4) both SEDLmRNA and sedlin proteins were expressed in both groups. The expression of SEDL mRNA in the experimental group was 0.79 卤0.12), which was significantly higher than that in the blank control group (0.11 卤0.04). The expression of sedlin protein in experimental group was 0.93 卤0.30). Compared with the blank control group (0.12 卤0.04), there was also a significant increase in p0.01. Conclusion 1) Recombinant adenovirus plasmid carrying SEDL gene was successfully constructed, and high titer recombinant adenovirus pAd5-SEDL was harvested. SEDL mRNA and sedlin protein were successfully expressed in hFOB1.19 cells. Recombinant adenovirus Ad5-SEDL carrying SEDL gene could significantly enhance the expression of SEDL mRNA and sedlin protein in hFOB1.19 cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

【相似文献】