青岛大学医学院学报2012，Vol.48Issue(2)：101-104,4.
利用SOE-PCR技术构建eglAp-rhIL-12融合基因的条件优化
OPTIMIZATION OF CONSTRUCTION OF FUSION GENE eglAp-rhIL-12 BY USING SOE-PCR TECHNIQUE
摘要
Abstract
Objective Using SOE-PCR technique, to link the promoter and signal pep tide sequence of Clostridium aceto-butylicum endo-l,4-glucanase gene (eglAp) and recombinant human IL-12 (rhIL-12) gene for construction of fusion gene eglAp-rhIL-12, and acquire repeatable SOE-PCR method by optimizing main conditions of SOE-PCR. Methods The eglAp sequence and rhIL-12 sequence were obtained by PCR amplification % the aim product was gained by adjusting the amount and ratio of the templates eglAp and rhIL-12 in SOE-PCR reaction system; the specificity of PCR product was improved by adjusting annealing temperature and primer amount. Results High purity and abundant PCR product could be obtained when SOE-PCR met the following conditions: in 25 μL PCR system, the amount of two templates eglAp and rhIL-12 was 5 - 10 ng, respectively, and mole ratio was 5 : 1. When annealing temperature was set at 71 ℃, with no primers were added, three cycles were amplified, when 0. 4 μL (10 μmol/L) primers were then added, and the annealing temperature was set at 61. 3 ℃ , 28 cycles amplified. Conclusion The key of getting a successful SOE-PCR is to properly adjust the ratio of templates mass and concentration, i. e. ratio being 1 : 1, concentration less than 5 ng (in 25 μL PCR system); if a high purity and abundant linking product is required, the annealing temperature should be appropriately lowered and the amount of primer be reduced.关键词
聚合酶链反应/基因融合/优化Key words
polymerase chain reaction/ gene fusion/ optimization分类
医药卫生引用本文复制引用
张艳丽,张文卿,徐腾飞,吕锐,刘颖..利用SOE-PCR技术构建eglAp-rhIL-12融合基因的条件优化[J].青岛大学医学院学报,2012,48(2):101-104,4.基金项目
山东省医药卫生科技发展计划重点项目(2009-HD005) （2009-HD005）
