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目的:构建重组腺病毒pAdEasy-GFP-GITRL,并测定病毒滴度。方法:从PIRES-GITRL载体上用BglⅡ和SalⅠ双酶切,回收目的基因GITRL,插入同样用BglⅡ和SalⅠ双酶切的穿梭质粒pAdtrack-CMV中,将线性化穿梭质粒骨架质粒pAdEasy-1共转染BJ5183菌,鉴定正确后,将重组腺病毒载体pAdEasy-GFP-GITRL转染HEK293细胞,以包装出完整腺病毒。TCID50法测定重组腺病毒滴度,Western blot法检测GITRL蛋白表达。结果:成功构建小鼠GITRL基因的重组腺病毒载体(pAdEasy-GFP-GITRL),经包装后获得高滴度表达GITRL基因的重组腺病毒,病毒滴度为2.0×109pfu/mL。Western blot结果显示重组病毒载体能表达GITRL蛋白。结论:重组腺病毒表达载体(pAdEasy-GFP-GITRL)的成功构建及重组腺病毒的获得为GITRL基因干预治疗奠定了基础。
Objective: To construct recombinant adenovirus pAdEasy-GFP-GITRL and determine the virus titer. METHODS: The target gene GITRL was digested with BglII and SalI from PIRES-GITRL vector. The shuttle plasmid pAdtrack-CMV also digested with BglII and SalI was inserted into pAdEasy-1 vector Staining BJ5183 bacteria, identified correctly, the recombinant adenovirus vector pAdEasy-GFP-GITRL transfected HEK293 cells to package the complete adenovirus. The recombinant adenovirus titers were determined by TCID50 method and GITRL protein by Western blot. Results: The recombinant adenoviral vector (pAdEasy-GFP-GITRL) of mouse GITRL gene was successfully constructed and the recombinant adenovirus expressing GITRL gene was obtained in high titer. The virus titer was 2.0 × 109pfu / mL. Western blot results showed that the recombinant viral vector can express GITRL protein. Conclusion: The successful construction of recombinant adenovirus expression vector (pAdEasy-GFP-GITRL) and the obtainment of recombinant adenovirus laid the foundation for the intervention of GITRL gene.