本文關鍵詞：SEDL基因重組腺病毒的構建及其對hFOB1.19細胞相關基因和蛋白表達的研究　出處：《重慶醫(yī)科大學》2012年碩士論文　論文類型：學位論文

  更多相關文章： 重組腺病毒 遲發(fā)性脊椎骨骺發(fā)育不良 SEDL 轉染 hFOB1.19細胞

【摘要】：目的 構建含SEDL基因的重組腺病毒質粒，收獲高滴度的重組腺病毒。采用SEDL基因重組腺病毒增強人SV40轉染成骨細胞（hFOB1.19）中SEDL mRNA及sedlin蛋白的表達，為探討SEDL基因及sedlin蛋白在骨骼代謝中的作用奠定基礎。 方法 采用定向克隆的方法將SEDL基因插入穿梭質粒pAdtrace-TO4，酶切及基因測序鑒定后命名為pAdtrace-SEDL；然后采用同源重組方法將pAdtrace-SEDL與腺病毒骨架質粒pAdEasy-l連接，構建重組腺病毒質粒pAd5-SEDL。酶切鑒定pAd5-SEDL后，用PolyJet將其導入HEK293包裝細胞，乒乓感染后獲得高滴度的重組腺病毒pAd5-SEDL。應用Ad5-SEDL感染hFOB1.19細胞，實驗分兩組：實驗組（hFOB1.19/Ad5-SEDL）、空白對照組（hFOB1.19）。通過MTT法檢測hFOB1.19細胞增殖；RT-PCR方法分別檢測hFOB1.19細胞內SEDLmRNA表達；Western Blot法檢測hFOB1.19細胞內sedlin蛋白表達。 結果 （1）酶切及基因測序證實重組腺病毒質粒pAd5-SEDL構建成功。(2)熒光顯微鏡證實重組腺病毒pAd5-SEDL成功導入HEK293包裝細胞。（3）收獲高滴度重組腺病毒pAd5-SEDL，，病毒滴度約為4.3×l011pfu/ml。（4）兩組細胞中均有SEDLmRNA及sedlin蛋白的表達。（5）實驗組SEDL mRNA表達量為（0.79±0.12）與空白對照組（0.11±0.04）相比明顯增加，差異有統(tǒng)計學意義（p0.01）。（6）實驗組sedlin蛋白表達量為（0.93±0.30），與空白對照組（0.12±0.04）相比也明顯增加（p0.01）。 結論 （1）成功構建了攜帶SEDL基因的重組腺病毒質粒，收獲高滴度重組腺病毒pAd5-SEDL，并成功感染hFOB1.19細胞。（2）hFOB1.19細胞表達SEDL mRNA及sedlin蛋白。（3）攜帶SEDL基因的重組腺病毒Ad5-SEDL可明顯增強hFOB1.19細胞SEDL mRNA及sedlin蛋白的表達。
[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 鹵0.12), which was significantly higher than that in the blank control group (0.11 鹵0.04). The expression of sedlin protein in experimental group was 0.93 鹵0.30). Compared with the blank control group (0.12 鹵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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R373

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