人PRKAG2全长基因的体外拼接及TA克隆载体的构建

Gene Splicing and Construction of PRKAG2 Gene TA Cloning Vector

目的:应用体外基因拼接及TA克隆技术构建含PRKAG2基因的载体。方法:通过商业途径购得PRKAG2基因的一个转录变体cDNA质粒(GenBank登记号BC068598),其在N端缺失了44个氨基酸,根据已知的PRKAG2基因序列(GenBank登记号NM_016203),设计搭桥引物及序列扩增引物,通过PCR搭桥方法,合成完整全序列的PRKAG2基因,并将其克隆到TA载体,将得到的阳性克隆测序鉴定。结果:拼接出全长1759bp的PRKAG2基因,目的基因连接到载体后测序与设计的PRKAG2基因完全一致。结论:成功的构建了全长人PRKAG2基因的TA克隆,为进一步研究PRKAG2基因的功能提供了模板。

Objective： To obtain the complete sequence of PRKAG2 by gene splicing and construct the recombining vector of PRKAG2 gene by TA cloning technique. Methods： The incomplete PRKAG2 cDNA clone （GenBank accession number： BC068598）, which lost 44 amino acids in N-end, was obtained by purchase. The PRKAG2 gene sequence was designed according to the known PRKAG2 gene sequence （GenBank accession number： NM_016203）. The full-length PRKAG2 gene was acquired using the designed primers by PCR-based gene assembly method. The PCR product was inserted into TA cloning vector. Results： The full-length PRKAG2 gene was obtained. The TA cloning vector was identified with the restriction enzyme cutting and sequencing. Conclusion： The full-length PRKAG2 gene TA cloning vector was constructed successfully.