LUAN Yi, YU Xiu-Ping, ZHAO Wei-Meng, GU Ji-Hui and ZHOU Ya-Bin. Human papillomavirus type 16 E6 gene's T-A cloning and expression in insect cell[J]. Virologica Sinica, 1998, 13(4).
Citation: LUAN Yi, YU Xiu-Ping, ZHAO Wei-Meng, GU Ji-Hui, ZHOU Ya-Bin. Human papillomavirus type 16 E6 gene's T-A cloning and expression in insect cell .VIROLOGICA SINICA, 1998, 13(4) : 305.

人乳头瘤病毒16型E_6基因的T-A克隆及在真核细胞中的表达

  • 由于HPV16E6蛋白能诱导机体保护性免疫反应,可作为基因治疗的靶抗原。用杆状病毒昆虫细胞表达系统制备了HPV16E6基因工程蛋白,拟用于宫颈癌细胞系小鼠模型抗癌的免疫治疗。用PCR技术从HPV16基因组中扩增获得转化基因E6的完整ORF,按TA策略将其克隆到自行制备的杆状病毒转移载体pVL1393T尾载体中,置于杆状病毒AcMNPVPolh晚期启动子控制之下,用此重组转移质粒pVL1393E6与杆状病毒DNA共转染昆虫细胞Sf9,经噬斑筛选获得带有编码E6蛋白基因的重组杆状病毒株,并在昆虫细胞Sf9中表达为非融合性E6蛋白。SDSPAGE电泳分析其分子量约为18kD,免疫印迹实验表明,此重组蛋白能被兔抗HPV16E6抗体所识别。

Human papillomavirus type 16 E6 gene's T-A cloning and expression in insect cell

  • The baculovirus insect cell expression system was used to prepare the HPV16 E 6 recombinant protein. The HPV16 E 6 complete ORF amplified by PCR from HPV16 genome was T A cloned into the baculovirus transfer vector pVL1393 T under AcMNPV Polh. This recombinant transfer plasmid pVL1393 E 6 DNA and baculovirus DNA were co transfected into insect cell Sf 9. After the plaque screening, the recombinant baculovirus AcMNPV E 6 was expressed in insect cells. The E 6, analysised by SDS PAGE, was 18kD, and was recognized by antibody against E 6 protein, which showed their natural antigenecity.

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    Human papillomavirus type 16 E6 gene's T-A cloning and expression in insect cell

    • 1. Department of Microbiology,Shandong Medical University

    Abstract: The baculovirus insect cell expression system was used to prepare the HPV16 E 6 recombinant protein. The HPV16 E 6 complete ORF amplified by PCR from HPV16 genome was T A cloned into the baculovirus transfer vector pVL1393 T under AcMNPV Polh. This recombinant transfer plasmid pVL1393 E 6 DNA and baculovirus DNA were co transfected into insect cell Sf 9. After the plaque screening, the recombinant baculovirus AcMNPV E 6 was expressed in insect cells. The E 6, analysised by SDS PAGE, was 18kD, and was recognized by antibody against E 6 protein, which showed their natural antigenecity.

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