体外连接法快速高效构建表达β-半乳糖苷酶的重组腺病毒载体

目的:采用体外连接法构建和制备重组腺病毒Adeno-X-LacZ,为构建具有治疗价值的重组腺病毒载体奠定基础。方法:PI-SceⅠ/I-CeuⅠ酶切pShuttle2-LacZ穿梭质粒,回收4.6 kb的LacZ基因表达盒,与经过相同酶切的Adeno-X病毒DNA连接,连接产物用SwaⅠ酶切,最终产物转化DH5α。重组质粒用PCR法和PI-SceⅠ/I-CeuⅠ酶切鉴定。pAdeno-X-LacZ用PacⅠ线性化后,用脂质体介导其转染至AD293细胞内包装扩增出重组腺病毒颗粒,采用CsC l密度梯度离心法纯化重组腺病毒Adeno-X-LacZ。采用X-gal染色观察Adeno-X-LacZ在AD293细胞内包装和HVSMC表达情况。结果:PCR扩增可见312 bp特异性条带,PI-SceⅠ/I-CeuⅠ酶切重组质粒后释放出4.6 kb LacZ基因表达盒。X-gal染色证实了在AD293细胞内成功扩增包装出重组腺病毒Ad-eno-X-LacZ和LacZ基因在HVSMC中得到有效表达。结论:体外连接法是一种快速、简便、高效的构建重组腺病毒质粒的方法,本研究为构建具有治疗价值的重组腺病毒奠定了基础,Adeno-X-LacZ为研究腺病毒介导的基因转移提供了一良好的对照载体。

A Rapid and Efficient Method for Constructing Recombinant Adenoviral Vector Expressing β-galactosidase by in Vitro Ligation

Objective To construct recombinant adenoviral vector expressing β-galactosidase by in vitro ligation and provide a basis for construction of recombinant adenovirus vector expressing therapeutic gene of interest.Methods pShuttle2-LacZ was digested with PI-Sce Ⅰ/I-Ceu Ⅰand 4.6 Kb fragment of LacZ gene expression cassette was recovered.This fragment was ligated to predigested Adeno-X viral DNA with PI-Sce Ⅰ/I-Ceu Ⅰ.The ligated product was digested with Swa Ⅰ.The resultant DNA was transformed into E.Coli.DH5α.The correct recombinant plasmid,pAdeno-X-LacZ,was identified by PCR and PI-Sce Ⅰ/I-Ceu Ⅰdigestion.The Pac I-digested,linearized pAdeno-X-LacZ was transfected into AD293 cells by Lipofectamine.Recombinant adenovirus,Adeno-X-LacZ,was purified with CsCl density gradient ultracentrifugation.HVSMC was infected with Adeno-X-LacZ.X-gal staining was performed to monitor the expression of β-galactosidase gene.Results There was a specific band of 312bp when pAdeno-X-LacZ was amplified by PCR.PI-SceⅠ/I-CeuⅠdigestion of pAdeno-X-LacZ released 4.6Kb of LacZ gene fragment.X-gal staining confirmed Adeno-X-LacZ was packaged successfully within AD293 cells and the expression of β-galactosidase gene in HVSMC.Conclusion In vitro ligation is a simple,rapid and efficient method for constructing recombinant adenoviral vector.This study provides a basis for construction of recombinant adenoviral vector carrying therapeutic gene of interest,Adeno-X-LacZ is also a useful control vector for the research of gene transfer mediated by recombinant adenovirus.