原核增强子样序列筛选及其功能区域鉴定  

Screening of prokaryotic enhancer-like sequences and identification of its domains

作　　者：崔成成[1] 王应明[1] 毕艳红[1] 李攀[1] 杨思达[1] 黄芬[1] 曾韦锟[1] 井申荣[1] 

机构地区：[1]昆明理工大学医学院,云南昆明650500

出　　处：《中国生物制品学杂志》2015年第3期251-256,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(31360619;31160193);云南省应用基础研究面上项目(2010ZC055;2012FB135)

摘　　要：目的从污水和土壤中富集的微生物菌体中筛选原核增强子样序列,构建包含增强子样序列的表达载体,并通过构建缺失突变体鉴定其功能区域。方法将人乳头瘤病毒（human papilloma virus,HPV）主要衣壳蛋白基因L1截短序列L11与氯霉素乙酰转移酶基因（chloramphenicol acetyltransferase,CAT）连接,作为报告基因,从污水和土壤富集的微生物菌体中筛选具有增强子活性的序列,构建包含增强子样序列的表达载体,表达HPV L11蛋白,检测其增强活性;通过构建ER1的缺失突变体,测定不同突变体对L11蛋白表达的作用情况,确定其功能区域。结果从样品基因组DNA中筛选出1条具有一定增强蛋白表达功能的增强子样序列,可使检测菌株氯霉素抗性提高5倍,HPV L11蛋白表达水平提高1.78倍。其增强活性主要位于117~317 bp区域。结论从污水和土壤中成功筛选到1条增强子样序列,携带有增强子样序列的表达载体可提高目的蛋白表达水平。Objective To screen prokaryotic enhancer-like sequences from the microorgans enriched in collected sewage and soil samples to construct expression vectors, and identify its domains by construction of depletion mutant. Methods The major capsid protein gene L1（named as L11）of truncated human papillomavirus（HPV）was linked to chloramphenicol acetyltransferase（CAT） gene, and used as a reporter gene for screening of sequences with activity of enhancer from the mi croorgans enriched in collected sewage and soil samples, based on which the expression vector containing enhancer-like sequences was constructed, and the expressed HPV L11 protein was determined for enhancing activity. The depletion mutant of ER1 was constructed, by which the effect of various mutants on expression of L11 protein was evaluated to de termine the domains. Results An enhancer-like sequence was screened from the samples, which increased the resistance of bacterial strain to chloramphenicol by 5 folds; and the expression level of HPV L11 protein by 1. 78 folds. The activity-enhancing region was located in 117 ~ 317 bp. Conclusion An enhancer-like sequences was successfully screened from the collected sewage and soil samples, and the expression vectors carrying the sequences increased the ex-pression level of target protein.

关 键 词：增强子样序列 筛选 外源基因 蛋白表达 功能区域 

分 类 号：Q786[生物学—分子生物学]