PTEN重组腺病毒载体的构建及其在气道平滑肌细胞的表达

Construction of a recombinant adenovirus vector containing PTEN and its expression in airway smooth muscle cells

目的应用pAdxsi载体系统构建携带PTEN基因的腺病毒载体。方法将PTENcDNA自载体pcDNA3-PTEN中切出,克隆至穿梭质粒pShuttle-GFP-CMV中,然后与腺病毒质粒pAdxsi在化学感受态细胞DH5α菌株内同源重组,经Pac-Ⅰ线性化后,重组子通过脂质体2000介导转染HEK293细胞,产生有感染能力的腺病毒,并测定其滴度,通过荧光显微镜能观察HEK293细胞内绿色荧光的表达;PCR检测腺病毒载体PTEN mRNA的表达,Western blotting检测PTEN蛋白在气道平滑肌细胞的表达。结果感染腺病毒载体的HEK293细胞表达GFP随着时间逐渐增强,并且出现明显的细胞病变效应,经过3轮扩增后病毒达到合适的滴度,构建的Ad-PTEN腺病毒滴度为2×1010pfu/ml,PCR扩增后经琼脂糖凝胶电泳证实其表达,Western blotting检测到腺病毒感染的气道平滑肌细胞内PTEN蛋白表达明显增高。结论成功构建了携带野生型PTEN腺病毒载体,为进一步研究PTEN基因应用于哮喘治疗提供了实验基础。

Objective To construct a recombinant adenovirus vector containing phosphatase and tensin homolog deleted on chromosome 10 （PTEN） using the pAdxsi system. Methods PTEN cDNA from plasmid pcDNA3-PTEN was cloned into the shuttle plasmid p Shuttle-GFP-CMV. The shuttle vector was transformed into competent DH5α strain with the vector pAdxsi to achieve the homologous recombination. The recombinant construct was subsequently linearized with PacI and transfected into HEK293 cells via Lipofectamine 2000. The recombinant adenovirus particles were collected, and after titration, the recombinant adenovirus was traced by monitoring GFP expression under fluorescence microscope. The expression of PTEN mRNA and protein in the recombinant adenovirus vector and airway smooth muscle cells were detected by PCR and Western blotting, respectively. Results GFP was expressed in HEK293 cells infected by recombinant adenovirus, and the expression intensity increased gradually with the passage of time, with obvious cytopathic effect （CPE） noted in the cells. After 3 cycles of amplification, the titer ofadenovirus containing PTEN reached an appropriate level. The viral titer ofpAdxsi-GFP-PTEN was 2×10^10 pfu/ml, and PTEN mRNA expression was detected by PCR. The homologous protein expressed in the infected human airway smooth muscle cells significantly increased in comparison with that in the control cells. Conclusion The recombinant adenovirus containing PTEN is constructed successfully, which provides an experimental basis for studying the role ofPTEN gene in asthma therapy.