腺相关病毒与人α_1干扰素基因重组体的构建

Construction of a recombinant of human interferon alpha-1 gene and adeno-associated virus vector

目的 构建腺相关病毒载体 (pWP8A)与人α1 干扰素 (hIFNα1 )基因重组体。 方法 用多聚酶链反应 (PCR)方法扩增获得hIFNα1 基因 ;以T A克隆方法将PCR扩增hIFNα1 基因亚克隆到PCR2 1质粒中 ,产生PCR2 1-hIFNα1 ;用EcoRI酶切PCR2 1-hIFNα1 后回收hIFNα1 基因片断 ,将此片断连接到pWP8A的EcoRI位点上 ,构建重组体pWP8A-hIFNα1 ;pWP8A -hIFNα1 转化JM10 9大肠杆菌扩增 ,hIFNα1 基因经测序鉴定符合Genebank中hIFNα1 序列 ,并用BamHI酶和XbalI酶双酶切鉴定hIFNα1 基因插入方向。 结果 经PCR扩增获得 5 67bphIFNα1 基因 ;构建重组体pWP8A -hIFNα1 ,经测序证实pWP8A -hIFNα1 中的hIFNα1 与Genebank中hIFNα1 序列相同 ,用BamHI酶和XbalI酶双酶切鉴定hIFNα1 基因插入方向正确的重组体pWP8A -hIFNα1 。 结论 成功构建腺相关病毒载体 (pWP8A)与人α1 干扰素(hIFNα1 )基因的重组体。

Objective To construct a recombinant of adeno-associated virus vector (pWP8A) and human interferon alphal (hIFNα 1) gene. Methods PCR was performed to amplify hIFNα 1 gene from a plasmid containing whole fragment of hIFNα 1 gene.Product of the PCR was subcloned into PCR2.1 vector to generate a recombinant PCR2.1-hIFNα 1.PCR2.1-hIFNα 1 was cut with EcoRI to release a fragment of hIFNα 1 gene.The fragment was cloned into pWP8A at EcoR I site to generate pWP8A-hIFNα 1.hIFNα 1 gene of pWP8A-hIFNα 1 was confirmed by sequencing,and inserted direction of hIFNα 1 gene was identified with BamH I and Xbal I. Results The whole fragment of hIFNα 1 gene was got by PCR.hIFNα 1 gene of PCR2.1-hIFNα 1 was released by digested with EcoR I,and was cloned into pWP8A to generate pWP8A-hIFNα 1.hIFNα 1 gene of pWP8A-hIFNα 1 was confirmed by sequencing. Conclusion We have successfully constructed a recombinant (pWP8A-hIFNα 1) of pWP8A and hIFNα 1 gene.