EGFP和LmxlA双基因共表达重组腺病毒的构建及检测

Construction and Coexpression of Recombinant Adenovirus Containing EGFP and Lmx1A

目的:通过携带绿色荧光蛋白(EGFP)和转录因子Lmx1A(LIM homeobox transcription factor1-alpha)的双顺反子重组腺病毒的构建和包装,评价双顺反子构建方法的可行性,为进一步探讨转录因子Lmx1A在间充质干细胞分化为神经元细胞的潜能提供实验基础。方法:通过PCR方法获得增强型绿色荧光蛋白(EGFP)和转录因子Lmx1A的CDS区序列,将其构建到双顺反子表达质粒pcDNA3.0BA,通过PCR的方法获得双顺反子表达盒,并插入到腺病毒穿梭质粒pShuttleCMV中,经过与腺病毒骨架质粒pAdEasy-1的同源重组,获得阳性重组质粒pAd-EGFP-Lmx1A,并进一步在HEK293细胞中包装为腺病毒。用携带EGFP和Lmx1A基因的重组腺病毒感染人脐带间充质干细(human umbilical cord mesenchymal stem cells,hUC-MSCs),通过RT-PCR、免疫荧光及Western blot的方法检测EGFP和Lmx1A的表达及产物定位。结果:透射电镜检查包装好的重组腺病毒为典型的腺病毒颗粒形态,大小约为75nm。Ad-EGFP-Lmx1A感染hUC-MSCs后检测到EGFP和Lmx1A的转录产物,表达产物互不影响,表达强弱无明显差异。结论:利用双顺反子真核表达质粒pcDNA3.0BA能够构建同时表达两个基因的重组腺病毒,腺病毒感染细胞后能够将两个基因传递至间充质干细胞中并表达。通过EGFP的观察可对腺病毒的表达效率进行实时观察,这些研究结果为任意组合的双基因表达建立了快速有效的构建方法,重组腺病毒Ad-EGFPLmx1A的成功构建也为将来研究Lmx1A在间充质干细胞分化为神经元过程中的作用提供了实验基础。

Object： The bicistronic recombinant adenovirns containing enhanced green fluorescent protein （EGFP） and transcription factors LmxlA （LIM homeobox transcription factor 1-alpha） was constructed and packaged. The feasibility of dicistronic construction methods was evaluated. An experimental basis provides for differentiation potential of mechymal stem cells into neurons. Methods： The CDS of EGFP of LmxlA, which was obtained by PCR, was inserted into the bicistronic expression plasmid pcDNA3.0BA. The dicistronic expression cassette which obtained by PCR, was inserted into the adenovirus shuttle plasmid pShuttle-CMV and followed homologous recombination with backbone plasmid pAdEasy-1. The adenovirus Ad-EGFP-LmxIA was packaged in HEK293 cells. Human umbilical cord mesenchymal stem ceils （hUC-MSCs） were infected with Ad-EGFP- Lmxl A. The expression and location of LmxlA and EGFP was identified by RT-PCR, immunofluorescence and Western blot technology. Results： The packaged adenovirus showed typical morphology of an adenoviral particle, about 75nm, through TEM examination. The expression product s of EGFP and LmxlA could be detected independently of each other. There was no significant difference in the expression strength. In conclusion, two genes can be expressed simultaneously in bicistronic eukaryotic expression plasmid pcDNA3.0BA. Two genes were transmitted into mesenchymal stem cells through adenovirus infection. Adenoviral expression efficiency can be observed in real time by observing the EGFP. These findings establish a rapid and effective method for constructing any two-gene expression. The construction of Ad-EGFP-LmxlA provides an important basis for the role exploration in mesenchymal stem cells differentiating into neurons.