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

作　　者：张严[1] 龚爱华[1] 金洁[1] 邵根宝[1] 彭琬昕[1] 

机构地区：[1]江苏大学基础医学与医学技术学院,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2011年第5期390-393,401,共5页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(30701031);江苏省高校自然科学基金资助项目(07KJB310018);江苏大学高级专业人才科研启动基金项目(09JDG072)

摘　　要：目的:利用体外DNA同源重组的方法分别构建含神经生长因子(NGF)和神经生长因子受体(TrkA)基因的真核表达载体。方法:在引物5'加上一段与载体克隆位点两端碱基序列相同的序列,PCR扩增目的基因NGF和TrkA,线性载体片段和目的基因片段经T4 DNA聚合酶的外切产生互补的单链DNA,然后37℃退火实现体外同源重组,转化并鉴定;将重组质粒转染293A细胞,免疫印迹鉴定目的基因的表达情况。结果:成功构建真核载体pcDNA3.1-NGF和pcDNA3.1-TrkA,转染细胞后的表达产物相对分子质量分别是31×103和140×103。结论:与常规重组技术相比,体外DNA同源重组技术是一种高效的DNA重组方法,且不需考虑目的片段的限制性酶切位点。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 140×103 and NGF was 31×103.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 T4DNA聚合酶 

分 类 号：R393[医药卫生—基础医学]