人CXCR4基因启动子萤光素酶报告载体的构建及鉴定

Construction and identification of a luciferase reporter vector containing human CXCR4 promoter

目的构建并鉴定人CXCR4基因启动子萤光素酶报告载体,为AIDS的靶向基因治疗奠定基础。方法利用PCR技术扩增人CXCR4基因启动子的核心片段,克隆至含新霉素抗性的萤光素酶表达载体pGL3-neo,构建含CXCR4启动子的萤光素酶表达载体pGL3-neo-CXCR4,重组子经双酶切、PCR及测序鉴定。再将构建的载体用脂质体转染体外培养的Jurkat细胞株,检测萤光素酶的表达活性。结果扩增得到CXCR4基因启动子序列,克隆入pGL3-neo-CXCR4的CXCR4基因启动子序列与GenBank报道的一致,实验组(pGL3-neo-CXCR4组)萤光素酶的表达活性为869 159.0±62 618.8,与空白组(未转染组)的464.2±44.0和对照组(pGL3-neo组)的39 088.4±3867.9相比,差异有统计学意义(F=1415.124,P均<0.05)。结论成功构建了含人CXCR4基因启动子的萤光素酶报告基因表达载体pGL3-neo-CXCR4。

Objective To construct and identify a luciferase reporter vector pGL3-neo-CXCR4 containing human CXCR4 promoter. Methods The gene Human promoter of CXCR4 was obtained by PCR amplification and was inserted into the rebuilded reporter vector pGL3-neo, the recombinant was identified by DNA sequencing, PCR and restriction enzyme digestion. The plasmid pGL4-CXCR4 was transfected into Jurkat cells, the luciferase activity in stable transfected cells was detected. Results CXCR4 promoter gene fragment was amplified by PCR. The insertion sequence of CXCR4 promoter in pGL3-neo-CXCR4 was in conformity with the GenBank reports . The luciferase activity in the pGL3-neo-CXCR4 group was 869 159.0 ±62 618.8,which was significantly higher that in the pGL3-neo group （39 088.4 ±3 867.9） and the blank group （464.2 ± 44.0）, P 〈 0.05. Conclusion Luciferase reporter vector pGL3-neo-CXCR4 containing human CXCR4 promoter can be constructed successfully.