NF1微基因及突变体微基因的克隆与鉴定  

Cloning and Identification of Minor Genes of NF1 and Its Mutants

作　　者：姚林林 李珺[1,2] 赵宏宇 蔡禄[1,2,3] Yao Linlin;Li Jun;Zhao Hongyu;Cai Lu(The Institute of Bioengineering and Technology, Inner Mongolia University of Science and Technology, Baotou, 014010;School of Life Science and Technology, Inner Mongolia University of Science and Technology, Baotou, 014010;Inner Mongolia Key Laboratory of Biomass-Energy Conversion, Baotou, 014010)

机构地区：[1]内蒙古科技大学生物工程与技术研究所,包头014010 [2]内蒙古科技大学生命科学与技术学院,包头014010 [3]内蒙古自治区生物质能源化利用重点试验室,包头014010

出　　处：《基因组学与应用生物学》2018年第4期1832-1837,共6页Genomics and Applied Biology

基　　金：国家自然科学基金(61271448;61671256;61662055);内蒙古自然科学基金(2014MS0311)共同资助

摘　　要：Ⅰ型神经纤维瘤病（Neurofibromatosis typeⅠ,NF1）是一种常染色体的显性遗传病,其致病性是由NF1基因突变引起。NF1基因m RNA前体受可变剪接的影响,NF1微基因是研究其可变剪接机制的重要工具。本研究从人类外周血中提取基因组DNA,并设计出克隆NF1及突变目的片段的两对引物,利用PCR方法扩增目的片段并导入p EGFP质粒载体,从而构建了分别包含NF1微基因及突变体微基因的载体p EGFP-NF1和p EGFP-NF1m。分别用Bam HⅠ和HindⅢ对NF1微基因及突变体微基因进行双酶切鉴定,均得到预期片段,测序结果正确,表明成功构建了NF1微基因及突变体微基因载体。用转染试剂将NF1微基因和突变体微基因的质粒转染到Hela细胞中,荧光检测说明真核表达载体NF1微基因与突变体微基因成功转染到细胞中。本研究为后续NF1的m RNA前体可变剪接以及调控NF1剪接因子的研究提供了参考。Neurofibromatosis typeⅠ（NF1） is an autosomal dominant hereditary disease caused by the mutation of NF1 gene. The pre-m RNA of NF1 gene is influenced by alternative splicing. Minor gene of NF1 is an important tool to study its mechanism of alternative splicing. In this study, the genomic DNA was extracted from human peripheral blood. Two pairs of primers for cloned NF1 and mutated fragmant were designed. The target fragments were amplified by the method of PCR and was inserted into the plasmid vector of p EGFP to construct vectors p EGFP-NF1 and p EGFP-NF1 m including minor genes of NF1 and its mutant, respectively. The anticipated fragments could be obtained from double enzyme digestion of NF1 and its mutant minor genes by Bam H Ⅰ and Hind Ⅲ, respectively. The sequencing results were correct, which showed that NF1 and mutant minor gene vectors were successfully constructed. NF1 and mutant minor gene plasmids were transfected into Hela cells with transfection reagent. The fluorescence detection showed that the eukaryotic expression vectors of NF1 and mutant minor genes were successfully transfected into the cells. The study might lay the foundation for further study on pre-m RNA alternative splicing of NF1 and the regulation of its splicing factors.

关 键 词：可变剪接 NF1基因 微基因 

分 类 号：Q78[生物学—分子生物学] R596.1[医药卫生—内科学]