江苏大学学报（医学版）2011，Vol.21Issue(5)：390-393,401,5.
应用体外DNA同源重组技术构建pcDNA3.1-NGF和pcDNA3.1-TrkA
Application of homologous recombination in vitro to construct pcDNA3.1-NGF and pcDNA3.1-TrkA
摘要
Abstract
Objective: In this study, we use the DNA homologous recombination in vitro to construct NGF and tyrosine kinase A (TrkA) eukaryotic expression vector. Methods: PCR primers for inserts were designed to contain appropriate 5' extension sequences. For the cloning reaction we generated the vector by cleavage with double restriction enzyme and generated the inserts TrkA and NGF by PCR. We treated both the vector and the inserts with T4 DNA polymerase to generate overhangs, then incubated vector and insert to promote recombination, and transformed the products into E. Coli and identified. Transfect the recombination DNA into 293A and identify the gene expression. Results: The expression vector pcDNA3. 1-NGF and pcDNA3.1-TrkA were constructed successfully by homologous recombination. Western blot showed TrkA protein expression in infected 293A cells was 140 x 103 and NGF was 31 x 103. Conclusion: Compared with conventional recombination technology, homologous recombination in vitro was a kind of high efficient DNA recombination method, and with no need for considering the restriction enzymes' site.关键词
体外DNA/同源重组/TrkA/神经生长因子/T4 DNA/聚合酶Key words
homologous recombination in vitro/ tyrosine kinase A/ nerve growth factor/ T4 DNA polymerase分类
医药卫生引用本文复制引用
张严,龚爱华,金洁,邵根宝,彭琬昕..应用体外DNA同源重组技术构建pcDNA3.1-NGF和pcDNA3.1-TrkA[J].江苏大学学报（医学版）,2011,21(5):390-393,401,5.基金项目
国家自然科学基金资助项目(30701031) （30701031）
江苏省高校自然科学基金资助项目(07KJB310018) （07KJB310018）
江苏大学高级专业人才科研启动基金项目(09JDG072) （09JDG072）
