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目的 :构建一种廉价高效的防治宫颈癌的人乳头瘤病毒新型双启动子DNA疫苗。方法 :设计合成细菌厌氧诱导启动子Nir ,含有FNR顺式反应元件、RBS和真核基因Kozak元件 ,插入pTriEx真核启动子CMVIE下游 ,构建pCMVnir双启动子载体。将人乳头瘤病毒 16型L1L7,融合基因 ,插入pCMVnir,制备pCMVnirL1E7,转化减毒沙门氏细菌SL32 6 1,厌氧诱导表达 ,SDS PAGE和Westernblot分析检测Nir启动子的活性。结果 :经DNA序列和限制性内切酶鉴定分析 ,成功构建了HPV双启动子DNA疫苗载体pCMVnirL1E7,转化沙门氏细菌SL32 6 1,厌氧条件下可诱导表达HPVL1E7蛋白质。结论 :成功构建了一种与胞内寄生细菌(减毒沙门氏细菌 )匹配的双启动子DNA疫苗新型载体 ,并构建了HPV16pCMVnirL1E7新型DNA疫苗
Objective: To construct a cheap and efficient DNA vaccine against human papillomavirus (HPV) double promoter for cervical cancer. Methods: The bacterial anaerobic induction promoter Nir was designed and synthesized, containing the FNR cis-response element, RBS and eukaryotic gene Kozak element, and inserted into the downstream of pTriEx eukaryotic promoter CMVIE to construct pCMVnir double promoter vector. The human papillomavirus type 16 L1L7 fusion gene was inserted into pCMVnir to prepare pCMVnirL1E7, and the transformed attenuated Salmonella SL3261 was transformed into anaerobic-induced expression. The activity of Nir promoter was detected by SDS PAGE and Western blot analysis. Results: DNA double-promoter DNA vaccine vector pCMVnirL1E7 was successfully constructed and transformed into Salmonella SL32 6 1. The expression of HPV L1 E7 protein was induced under anaerobic conditions by DNA sequence and restriction endonuclease analysis. CONCLUSION: A novel double-promoter DNA vaccine matched with intracellular parasitic bacteria (attenuated Salmonella enterica) was successfully constructed and a novel DNA vaccine of HPV16pCMVnirL1E7