摘要
目的构建表达人类晶状体来源的上皮生长因子p52(lens epithelium derivedgrowthfactor p52,LEDGFp52)重组腺病毒载体,检测LEDGFp52重组腺病毒能否在真核细胞中有效表达目的基因。方法将LEDGFp52基因片段亚克隆到腺病毒穿梭质粒pAdTrack-CMV上构建腺病毒穿梭载体pAdTrack-CMV-LEDGFp52,将腺病毒穿梭载体pAdTrack-CMV-LEDGFp52与5型腺病毒骨架质粒pAdEasy-1共转染BJ5183细菌,经细菌内同源重组产生携带LEDGFp52基因的重组腺病毒载体pAd-LEDGFp52,将pAd-LEDGFp52经脂质体法转化293细胞包装产生重组腺病毒Ad-LEDGFp52,将Ad-LEDGFp52体外转染293细胞,CPE法及Westernblot观察目的基因的表达。结果构建了表达LEDGFp52基因的重组腺病毒质粒,E.coli内成功同源重组腺病毒,在293细胞内包装产生重组腺病毒Ad-LEDGFp52,滴度可达5×1012PFU·L-1。在真核细胞中有效表达目的基因LEDGFp52,出现显著的CPE效应。结论成功构建表达LEDGFp52的重组腺病毒载体,为进一步进行LEDGF p52基因功能的研究提供了实验基础。
Objective To construct recombinant adenovtrus vector carrying LEDGFp52 gene by homologous recombination In bacteria and to detect Its expression In vitro. Methods LEDGFp52 gene was cloned to adenovtral shuttle plasmtd pAdTrack-CMV. Then the resultant pAdTrack-CMV-LEDGFp52 was cotransfected Into BJ5183 bacteria with the adenovtrus backbone plasmtd pAdEasy-1. The adenovtrus plasmtd carrying LEDGFp52 was generated with homologous recombination in bacteria and the adenovtruses were produced in 293 cells. 293 cells were infected with adenoviruses and the expression of LEDGFp52 was detected by CPE（cytopathic effect） and westernblot. Results The titer of Ad-LEDGFp52 adenoviruses was up to 5 × 10^12 PFU· L^-1 after proliferation in 293 cells. LEDGFp52 was expressed efficiently in 293 cells after infection. Conclusion The recombinant adenoviruses expressing LEDGF p62 was constructed successfully and could be used further in gene transfection experiments.
出处
《眼科新进展》
CAS
2006年第12期900-903,共4页
Recent Advances in Ophthalmology
