重叠延伸PCR法在合成人骨保护素N端编码序列中的应用  

Amplification of DNA sequence Encoding N-terminal Fragment of Osteoprotegerin Using Overlap-extension PCR

作　　者：王宝利[1] 梁晖[1] 戴芳[1] 郭刚[1] 张镜宇[1] 

机构地区：[1]天津医科大学内分泌研究所二室,天津300070

出　　处：《天津医科大学学报》2004年第3期343-345,共3页Journal of Tianjin Medical University

基　　金：天津市自然科学基金资助项目 (编号 :043606811)

摘　　要：目的 :获得人骨保护素 (OPG)N端D1～D4结构域编码序列。方法 :OPGN端D1～D4域仅由2个外显子编码。采用改良方法自人血标本提取基因组DNA ,以此DNA作为模板 ,PCR分别扩增OPG基因外显子2和外显子3 ,并使外显子2的3'引物和外显子3的5'引物具有相互重叠的一段序列。以2种PCR产物的混合物作为模板 ,采用重叠延伸法再次进行PCR ,扩增产物克隆于载体pGEM -Teasy 进行序列分析。结果 :PCR扩增得到N端编码序列 ,序列分析证实该序列完全正确。结论 :重叠延伸PCR法合成人OPGN端编码序列的完成为基因工程研制有生物活性蛋白提供了可行的技术手段。Objective:To amplify the coding sequence for N-terminal fragment of Osteoproˉtegerin.Methods:The N-terminal D1～D4region of OPG is encoded by only two exons,exon2and exon3.In the present study,we amplified the sequence coding for this region by ampliˉfying the two exons respectively,and then linking them through a so-called overlap-extenˉsion method.In detail,as designed,the antisense primer for exon2had a sequence of20bp that was homogeneous with the sense primer for exon3.The PCR products for exon2and3were mixed as template,and the sense primer for exon2and antisense primer for exon3were used together to initiate the synthesis of the coding sequence for D1～D4region.The amplified fragment was finally cloned into pGEM-T easy and then sequenced.Results:As desired,the fragment of interest was obtained.The sequencing confirmed that its sequence was correct.Conclusion:It provides a necessary basis for the preparation of bioactive D1～D4domain of osteoprotegerin by genetic engineering.

关 键 词：重叠延伸 聚合酶链式反应 骨保护素 

分 类 号：R589.5[医药卫生—内分泌]