重组hTERT腺病毒载体的构建、包装与纯化及其意义

Effects of construction of hTERT-Ad and the adenovirus packaging

目的利用分子生物技术构建人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)基因腺病毒表达载体并检测hTERT基因腺病毒转染细胞的功能表达。方法用凝胶电泳、测序、Western blot-ting等鉴定hTERT质粒。用PCR技术扩增hTERT基因,构建hTERT-PIRES2-EGFP基因载体,重组到pAd腺病毒,转染293A细胞扩增病毒。终点稀释分析法测病毒滴度。CsCl连续梯度离心纯化。结果凝胶电泳、测序和Western blotting检测hTERT基因分子量、序列以及功能是正确的。并且成功构建hTERT腺病毒,病毒浓度达2.1×1012ifu/mL。结论成功构建hTERT腺病毒,为hTERT基因治疗的基础研究和动物实验研究奠定了基础。

【Objective】 To construct the hTERT-Ad and identify the function of the hTERT-Ad.【Methods】 Amplify hTERT gene plasmid with molecular cloning technology.Identify the success of hTERT gene plasmid amplification with gel electrophoresis,sequencing and Western Blot technique after the plasmid transfect into cells.Amplify hTERT gene with designing special primers and PCR technology,then link to the hTERT-PIRES2-EGFP carrier,then recombinate gene to adenovirus vector and prepared for adenovirus packaging.Amplify virus in 293A cells,measure virus titre with end dilution assay and purify the amplified virus with continuous gradient centrifugation in the CsCl cation column.【Results】 It is correct when identify the hTERT gene plasmid by gel electrophoresis,sequencing and Western Blot technique.The hTERT-Ad was constructed successfully and the virus titre was 2.1×1012 ifu/mL.【Conclusions】 The successful construction of the hTERT-Ad is essential for the basic research of hTERT gene therapy.