人蛋白激酶CβⅡ基因开放读码框的克隆及序列分析

Cloning and sequencing analysis of human PKCβⅡgene ORF

目的:克隆人蛋白激酶CβⅡ基因(PRKCB1)的开放读码框(Open reading frame,ORF),为其进一步的功能研究提供基础。方法:采用逆转录-巢式PCR法,从人脐静脉内皮细胞株中扩增出含PRKCB1基因ORF全长的片段,所得片段经加"A"处理并插入T载体。经蓝白筛选,用特异引物扩增阳性重组子,得到编码人蛋白激酶CβⅡ(Protein kinase CβⅡ,PKCβⅡ)基因的ORF,并与T载体相连,测序鉴定。结果:通过巢式RT-PCR及T/A克隆获取了PRKCB1基因的ORF,测序结果与GenBank报道序列一致。结论:成功克隆了PRKCB1基因的ORF,在技术路线上可以为某些难克隆的基因提供参考。

Objective：To clone human PRKCB1 gene open reading frame（ORF） in order to further research on it＇s function. Methods：RT-nested PCR method was adopted to the amplify the total length of human PRKCB10RF from human umbilical vein endothelial cell （HUVEC）.T vector was inserted into the harvested fragment after a tail was added. With blue white screening,the gene ORF encoding human PKCβⅡ was obtained by special primers amplifying recombinant plasmids,and linked into T vector.Then the plasmid was identified by sequencing. Results：The human PRKCB1 ORF was amplified successfully with RT-nested PCR and T/A cloning,and the gene sequence was completely consistent with that reported in GenBank. Conclusion：Human PRKCB1 gene ORF was successfully cloned. The strategy of cloning may provide technical references for some genes hard to be cloned.