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人乳头瘤病毒16型致瘤基因E6在昆虫细胞中表达及其蛋白抗原特性的初步研究 |
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栾怡 于修平 赵蔚明 贾继辉
【摘要】 目的 研究人乳头瘤病毒(HPV)相关肿瘤患者的HPV16 E6抗体水平及其流行病学意义；探讨用杆状病毒-昆虫细胞表达载体系统表达的HPV16 E6蛋白的抗原性。方法 用PCR从HPV16基因组中扩增出HPV16 E6完整基因，克隆至转移载体pVL1393中，重组质粒DNA与线性杆状病毒DNA共转染昆虫细胞Sf-9,经噬斑筛选获得带有编码E6基因的重组杆状病毒株，并在昆虫细胞中表达。结果 Western blot 和高效液相色谱法检测HPV16 E6表达蛋白，其分子量约为18000。免疫印迹检测显示其能与兔抗HPV16 E6多抗特异性结合。酶联免疫吸附试验表明此重组蛋白能被人HPV16阳性血清所识别。结论 昆虫细胞表达的HPV16 E6蛋白，具有良好的抗原性，可用于检测HPV16 E6特异性免疫球蛋白IgG和IgM抗体。
【主题词】 乳头瘤病毒，人 E6基因 杆状病毒 基因表达
Expression of human papillomavirus type 16 oncogene E6 in insect cells and the preliminary study of its immunological characteristics Luan Yi,Yu Xiuping,Zhao Weiming,et al.Department of Microbiology,Shandong Medical University,Ji nan 250012
【Abstract】 Objectives In order to study the antibody titer of HPV16 E6 in HPV associated tumor patients and its epidemiological significance. We have used the baculovirus expresss vector system to express HPV16 E6 ORF in insect cells,and primarily studied its antigenicity. Methods The complete E6 gene fragment has been amplified by PCR from HPV16 genome and cloned into the baculovirus transfer vector pVL1393.The insect cells Sf-9 were cotransfected with liner AcMNPV and recombinant vector DNA.After the plaque assay screening,the recombinant baculovirus was expressed in insect cells. Results We carried out Western blot and high performance liquid chromatography to detect the expressed products.To identify the 18kD protein as products of E6 ORF,these non-fusion proteins were recognized by rabbit antibodies against E6 protein and human HPV16 positive sera which showed their similar antigenity to natural protein.Conclusions HPV16 E6 proteins,expressed in insect cells, possess antigenicity similar to natural proteins.They appear to be a good antigen for the detection of HPV16 E6-specific immunoglobulin IgG and IgM antibodies.
【Subject words】 Human papillomavirus type 16 E6 protein Baculorvirus Gene expression
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