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

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

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。

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 Ⅰ and 4.6 Kb fragment of LacZ gene expression cassette was recovered. This fragment was ligated to predigested Adeno - X viral DNA with PI - SceⅠ/I - Ceu Ⅰ . The ligated product was digested with Swa Ⅰ . The resultant DNA was transformed into E. Coli. DHSα. The correct recombinant plasmid, pAdeno- X- LacZ ,was identified by PCR and PI - Sce Ⅰ/I - Ceu Ⅰ digestion. 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 - Sce Ⅰ/I - CeuⅠ digestion 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.