秦川牛Gli2基因重组干扰腺病毒的构建

Construction of Gli2 gene adenovirus interfering vector in Qinchuan cattle

【目的】构建秦川牛醇溶蛋白2(Gli2)基因的重组干扰腺病毒,为研究Hh信号通路在秦川牛脂肪形成过程中的功能奠定基础。【方法】构建3条短发卡RNA(shRNA):shRNA-LY1、shRNA-LY2、shRNA-LY3,利用双荧光素酶报告系统检测其干扰效率。再用干扰效率较高的shRNA-LY3构建腺病毒干扰载体pAD/PL-DEST/CMV-GFP/U6-LY3,转染HEK 293A细胞进行腺病毒的包装并扩繁以提高病毒滴度。利用LaSRT法测定腺病毒的滴度,并将得到的病毒以不同剂量侵染秦川牛脂肪细胞,确定其最佳感染复数(MOI)。【结果】筛选得到shRNA-LY3干扰效率最高,达到85%;成功构建了秦川牛Gli2基因腺病毒干扰载体pAD/PL-DEST/CMV-GFP/U6-LY3,包装后获得了重组干扰腺病毒Ad-shRNA-LY3;LaSRT法测得其滴度为1×10~9 PFU/mL。腺病毒对秦川牛脂肪细胞的最佳MOI为50。【结论】成功构建了秦川牛Gli2基因腺病毒干扰载体,包装后获得了高滴度的重组干扰腺病毒Ad-shRNA-LY3,其对秦川牛脂肪细胞的最佳MOI为50。

【Objective】The recombinant adenovirus of Qinchuan beef gliadin 2(Gli2)gene was constructed to lay a foundation for studying the function of Hh signaling pathway in fat formation of Qinchuan cattle.【Method】Three short hairpin RNAs(shRNAs)of shRNA-LY1,shRNA-LY2,and shRNA-LY3 were constructed and their interference efficiencies were screened using a dual luciferase reporter system.The adenovirus interfering vector pAD/PL-DEST/CMV-GFP/U6-LY3 was constructed with the highest interference efficiency.HEK 293 A cells were transfected and packaged with adenovirus to increase virus titers.LaSRT method was used to measure granularity.The titer of the virus,and the resulting virus was infected with Qinchuan cattle adipocytes at different doses to determine the optimal multiplicity of infection(MOI).【Result】The screening efficiency of shRNA-LY3 was up to 85%.Qinchuan cattle Gli2 gene adenovirus interfering vector pAD/PL-DEST/CMV-GFP/U6-LY3 was successfully constructed.After packaging,recombinant interfering adenovirus Ad-shRNA-LY3 was obtained.The titer of pAD/PL-DEST/CMV-GFP/U6-LY3 and LaSRT was 1×10~9 PFU/mL.The best MOI for adipocytes on bovine adipocytes was 50.【Conclusion】The Qinchuan cattle Gli2 gene adenovirus interfering vector was successfully constructed and the high titer of recombinant adenovirus was obtained after packaging.The best MOI for bovine adipocytes was 50.