国际流行病学传染病学杂志
國際流行病學傳染病學雜誌
국제류행병학전염병학잡지
INTERNATIONAL JOURNAL OF EPIDEMIOLOGY AND INFECTIOUS DISEASE
2013年
4期
232-235
,共4页
贾静%刘小利%颜冬梅%魏文鹏%王孝举
賈靜%劉小利%顏鼕梅%魏文鵬%王孝舉
가정%류소리%안동매%위문붕%왕효거
螺杆菌,幽门%REGⅠ α基因%重叠延伸PCR技术
螺桿菌,幽門%REGⅠ α基因%重疊延伸PCR技術
라간균,유문%REGⅠ α기인%중첩연신PCR기술
Helicobacter pylori%REG Ⅰ α gene%Overlap extension PCR
目的 采用重叠延伸PCR技术克隆人REG Ⅰα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REG Ⅰα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REGⅠ α基因cDNA,并构建REGⅠα基因真核表达载体.结果 扩增REGⅠ α基因Exon2～6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183 bp,Exon4 ～6连接产物318 bp;获得全长REG Ⅰα基因cDNA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-002909.4).结论 成功克隆人REGⅠ α基因cDNA,并构建真核表达载体,有助于为REGⅠ α基因参与胃癌发生的机制研究提供实验材料.
目的 採用重疊延伸PCR技術剋隆人REG Ⅰα基因,以便研究其在幽門螺桿菌感染促胃炎以及胃癌轉化的病理過程中的作用.方法 設計人REG Ⅰα基因2～6號外顯子OE-PCR引物,分段擴增各外顯子DNA片斷,通過重疊延伸PCR法將各外顯子逐一連接,穫得全長REGⅠ α基因cDNA,併構建REGⅠα基因真覈錶達載體.結果 擴增REGⅠ α基因Exon2～6號外顯子穫得的產物長度分彆為64bp、119bp、138bp、112bp和68bp;分彆連接穫得Exon2～3連接產物長度為183 bp,Exon4 ～6連接產物318 bp;穫得全長REG Ⅰα基因cDNA長度為501bp.所穫擴增產物序列與預期設計相符.插入到真覈載體pcDNA3.1的片斷大小符閤預期設計,測序結果與NCBI數據庫100％相符(NM-002909.4).結論 成功剋隆人REGⅠ α基因cDNA,併構建真覈錶達載體,有助于為REGⅠ α基因參與胃癌髮生的機製研究提供實驗材料.
목적 채용중첩연신PCR기술극륭인REG Ⅰα기인,이편연구기재유문라간균감염촉위염이급위암전화적병리과정중적작용.방법 설계인REG Ⅰα기인2～6호외현자OE-PCR인물,분단확증각외현자DNA편단,통과중첩연신PCR법장각외현자축일련접,획득전장REGⅠ α기인cDNA,병구건REGⅠα기인진핵표체재체.결과 확증REGⅠ α기인Exon2～6호외현자획득적산물장도분별위64bp、119bp、138bp、112bp화68bp;분별련접획득Exon2～3련접산물장도위183 bp,Exon4 ～6련접산물318 bp;획득전장REG Ⅰα기인cDNA장도위501bp.소획확증산물서렬여예기설계상부.삽입도진핵재체pcDNA3.1적편단대소부합예기설계,측서결과여NCBI수거고100％상부(NM-002909.4).결론 성공극륭인REGⅠ α기인cDNA,병구건진핵표체재체,유조우위REGⅠ α기인삼여위암발생적궤제연구제공실험재료.
Objective To obtain the full length cDNA of human REG Ⅰ α 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 REG Ⅰ α 2-6 exons were designed,and each exon was amplified by PCR.Full length cDNA of REG Ⅰ α was obtained by connecting all the exons using OE-PCR method.The eukaryotic expression system of REG Ⅰ α 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 Exont2-3 and Exon4-6 were 183 bp and 318 bp respectively.Finally the full length of obtained REG Ⅰ α 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 PEG Ⅰ α gene cDNA is cloned successfully and the expression vector is coustructed which could be used for the mechanism study of REG Ⅰ α in the gastric cancer pathogenesis.
