摘要
目的:构建含血红素氧化酶1基因的重组腺病毒及鉴定感染效率。方法:实验于2003-05/2004-06在解放军第三军医大学西南医院消化科实验室完成。①用限制性内切酶XhoⅠ+HindⅢ从克隆载体PRH01中切出血红素氧化酶1基因片段,亚克隆至Puc18中,将之用KpnⅠ+HindⅢ双酶切,再次亚克隆至质粒pAdTrack-巨细胞病毒中,形成转移质粒pAdTrack-Puc18-PRHO1。②将pAdTrack-Puc18-PRHO1PmeⅠ酶切线性化后与腺病毒基因组质粒pAdEasy-1共转化大肠杆菌BJ5183,得到含目的基因的重组体质粒。③重组腺病毒PacⅠ酶切后用脂质体转染293细胞,包装成重组体腺病毒AdH01。④对重组体腺病毒进行鉴定采用聚合酶链反应方法。结果:①利用氯化钙法由pAdTrack-Puc18-PRHO1和pAdEasy-1共转化BJ5183感受态菌,可获得阳性重组体细菌克隆。②重组腺病毒基因组DNA,用目的基因血红素氧化酶1引物进行聚合酶链反应检测,可特异性扩增出356bp的预期条带,而空载体组未能扩增出此片段,表明血红素氧化酶1基因已克隆入重组体腺病毒基因组中,根据绿色荧光蛋白记数法测得腺病毒滴度1.4×1013空斑形成单位/L。③重组腺病毒感染效率的测定结果:用浓缩后的重组腺病毒以感染复数为10感染肾小球内皮细胞,约85%的内皮细胞出现荧光,表明腺病毒AdH01在体外能有效表达相应的基因产物。结论:细菌内同源重组法获得AdH01在体外能有效表达。
AIM： To construct the recombinant adenovirus of heme oxygenase-1 and evaluate the infectinal efficiency.METHODS： This experiment was done from May 2003 to June 2004 in the Laboratory of Gastroenterology Research Center of Southwestern Hospital of Third Military Medical University of Chinese PLA. ① The segment of Heine oxygenase-1 gene was liberated from the cloning vector of PRHO-1 via restriction endomuclease of Xho Ⅰ ＋Hind Ⅲ, and subclone into Puc18 which was digested by Kpn Ⅰ ＋Hind Ⅲ double enzyme cutting and subclone into shuttle vector of plasmid pAdTrack-eytomegalovirus forming transfer plasmid of pAdTrack-Pucl8-PRHOl. ② After pAdTrack-Puc18-PRHO1 Pine Ⅰ enzyme cutting was linearized with adenovirus viral genome plasmid pAdEasy-1 cotransformed into bacillus coli of BJ5183 cells. ③ The recombination adenovirus： After the Pac Ⅰ enzyme cutting liposomes was used to transfer 293 cells, and packed into recombination adenovirus AdH01. ④ The recombination adenovirus was evaluated with polymerase chain reaction （PCR）. RESULTS： ① Positive recombinant bacterial clones were obtained after cotransformafion of BJ5183 competence bacteria with pAdTrack-Puc18-PRHO1 and pAdEasy-1 by method of calcium chloride. ② DNA in recombination adenovirus gene group,the PCR test was performed with objective gene heme oxygenase-1 primer could specific amplification the experctation strap of 356 bp, while this section did not amplified in zero load group, which indicated that the heme oxygenase-1 gene had cloned into recombinant adenovirus genome. The titre of adenovirus was 1.4×10^13 with nul-locus formation unit/L. ③ The result of determination of recombination adenovirus infection efficiency： The recombination adenovirus after coucentration at the infection complex numbor of 10 to infect endothelial cell of renal corpuscle, and about 85% of endothelial cell appeared fluorescence, which indicated that adenovirus could express effectively the corresponding gene product in vitro. CONCLUSION： The method of homologous recombination in bacteria can obtain AdH01 which can express effectwely in vitro.
出处
《中国临床康复》
CSCD
北大核心
2005年第27期70-71,共2页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金课题(30170436)~~
