重叠延伸聚合酶链反应法克隆人REGⅠα基因  

Cloning of hmnan REGⅠα gene by overlap extension polymerase chain reaction

作　　者：贾静[1] 刘小利[2] 颜冬梅[1] 魏文鹏[3] 王孝举[1] 

机构地区：[1]浙江省医学科学院分子医学中心,杭州310013 [2]浙江医院神经内科,杭州310013 [3]浙江省温州市温州医学院生命科学学院,325000

出　　处：《国际流行病学传染病学杂志》2013年第4期232-235,共4页International Journal of Epidemiology and Infectious Disease

基　　金：浙江省科技厅科技条件建设（2011F10015）;浙江省钱江人才计划（2011R10070）;浙江省自然科学基金青年基金（LQ12H16013）;浙江省医药卫生科技计划项目（2011KYB005）

摘　　要：目的采用重叠延伸PCR技术克隆人BEGⅠα基因，以便研究其在幽门螺杆菌感染促胃炎以及胃癌转化的病理过程中的作用。方法设计人BEGⅠα基因2．6号外显子OE-PCR引物，分段扩增各外显子DNA片断，通过重叠延伸PcR法将各外显子逐一连接，获得全长REGⅠα基因eDNA，并构建REGⅠα真核表达载体。结果扩增REGⅠα基因Exort2—6号外显子获得的产物长度分别为64bp、119bp、138bp、112bp和68bp；分别连接获得Exon2～3连接产物长度为183bp，Exon4～6连接产物318bp；获得全长BEGIa基因eDNA长度为501bp。所获扩增产物序列与预期设计相符。插入到真核载体pcDNA3．1的片断大小符合预期设计，测序结果与NCBI数据库100％相符（NM-（D2909．4）。结论成功克隆人BEG工a基因cDNA，并构建真核表达载体，有助于为REG工a基因参与胃癌发生的机制研究提供实验材料。Objective To obtain the full length cDNA of human REGⅠα gene using gene splicing by overlap extension PCR technique （OE-PCR） for further study its role in pathogenesis of gastric cancer from gastritis caused by Helicobacter pylori infection. Methods Specific OE-PCR primer pairs for REGⅠα 2-6 exons were designed, and each exon was amplified by PCR. Full length cDNA of REGⅠα was obtained by connecting all the exons using OE-PCR method. The eukaxyotic expression system of REGⅠα was constructed. Results The lengths of PCR amplified products of 2-6 Exons were 64 bp, 119 bp, 138 bp, 112 bp and 68 bp respectively. The lengths of connected products of Exon2- 3 and Exon4-6 were 183 bp and 318 bp respectively. Finally the full length of obtained REGⅠα cDNA was 501 bp. The sequences of all the amplified products matched to the predicted designs. The length of the sequence inserted in the pcDNA3.1 eukaryotic vector matched to the predicted designs and the sequencing result was 100% consistent with the NCBI data （NM-002909.4）. Conclusions Human REG I a gene cDNA is cloned successfully and the expression vector is constructed which could be used for the mechanism study of REGⅠα in the gastric cancer pathogenesis.

关 键 词：螺杆菌 幽门 BEGIa基因 重叠延伸PCR技术 

分 类 号：R318[医药卫生—生物医学工程] Q78[医药卫生—基础医学]