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目的:在已克隆的小鼠GATA3基因基础上构建腺病毒载体,制备GATA3-adv。方法:Ad-Easy法通过同源臂重组的方式在细菌中获得重组腺病毒基因组质粒。将克隆了外源基因的腺病毒穿梭质粒与携带了腺病毒大部分基因组的质粒共转化BJ5183细菌,在细菌重组酶的作用下经抗性筛选获得重组腺病毒基因组质粒,将其线性化后转染HEK293中获得有活性的包装腺病毒。腺病毒荧光检测试剂盒定量腺病毒浓度。结果:成功制备了小鼠Adv-GATA3,其浓度为3.34×109copies/ml。结论:小鼠GATA3基因重组腺病毒载体的成功构建为进一步在基因水平进行免疫紊乱性疾病的干预研究奠定了基础。
OBJECTIVE: To construct adenovirus vector based on the cloned mouse GATA3 gene and prepare GATA3-adv. Methods: Recombinant adenovirus plasmid was obtained from bacteria by Ad-Easy method by homologous arm recombination. The adenovirus shuttle plasmid which cloned exogenous gene was cotransformed with BJ5183 bacteria carrying plasmids of most of the adenovirus genome and the recombinant adenovirus plasmid was obtained through resistance screening under the action of bacterial recombinase and then linearized HEK293 infected with active packaging adenovirus. Adenovirus Fluorescence Detection Kit Quantify Adenovirus Concentrations. Results: Mouse Adv-GATA3 was successfully prepared at a concentration of 3.34 × 109 copies / ml. Conclusion: The successful construction of mouse GATA3 gene recombinant adenovirus vector lays a foundation for the further study on the intervention of immunological disorders at the genetic level.