应用E1区缺陷载体构建携带SEA基因的选择性腺病毒

Applied E1 region defective vector to construct selective adenovirus carrying SEA gene

目的:构建由端粒酶启动子(hTERT)调控的携带超抗原金黄色葡萄球菌肠毒素A(staphylococcus aureusenterotoxin A,SEA)基因的选择性复制重组腺病毒。方法:应用酶切、连接方法将hTERT连接于腺病毒穿梭质粒E1A和E1B序列之前调控E1A和E1B蛋白的表达,CMV启动子和SV40分别调控目的基因SEA的表达和终止,利用脂质体将腺病毒骨架质粒和穿梭质粒共同转染人胚肾293细胞(HEK-293)进行同源重组,获取选择性复制腺病毒CMV-SV40-hTERT(CSS-H),氯化铯梯度离心纯化重组腺病毒,并测定病毒滴度。结果:应用PCR验证、双酶切分析、序列测定等方法验证重组质粒基因片段连接正确无突变,通过同源重组获得携带超抗原SEA基因片段的CSS-H重组腺病毒,测定滴度为2.9×1010pfu/ml。结论:携带SEA基因选择性复制重组腺病毒CSS-H成功构建。

Objective: Constructing the adenovirus of selective replication carrying staphylococcus aureus enterotoxin A(SEA) gene control by the telomerase promoter(hTERT).Methods: Applied the method of cleavage and enzyme linked to connect the hTERT before E1A and E1B sequence of the adenovirus shuttle vector in order to control E1A and E1B protein expression.CMV promoter and SV40 respectively controled purpose gene expression and termination.Adenovirus shuttle vector and backbone plasmid co-transfected into human embryonic kidney 293 cells(HEK-293) by liposome and made homologous recombination,obtained selective replication adenovirus CMV-SV40-hTERT(CSS-H),cesium chloride gradient centrifugation purification restructuring adenovirus,and determined their tite.Results: The application of PCR validation,double enzyme histochemical cut analysis,sequence method validation restructuring plasmid connected correctly had not mutated gene fragments by homologous recombination,won the genetic sequences carry super antigen SEA the CSS-H restructuring adenovirus,the virus tite was 2.9×1010 pfu/ml.Conclusion: Selective replication adenovirus CSS-H Carrying SEA gene was successfully constructed.