牛γ-干扰素基因在大肠杆菌中的克隆

Cloning of bovine interferon-γ gene in Escherichia Coli

根据牛γ干扰素(bovIFN γ)基因的cDNA序列设计了一对引物。应用一步法逆转录聚合酶链式反应(RT PCR)技术,从奶牛脾脏淋巴细胞的总RNA中扩增出特异性片段,将RT PCR产物克隆到pMD18 T载体中,经过限制性酶切分析、测序证实扩增得到的bovIFN γ基因序列正确。将该基因片段切下并克隆到大肠杆菌表达载体pGEX 4T 1中,将pGEX 4T 1/bovIFN γ转化到大肠杆菌中DH5α中。

Bovine interferon-gamma (bovIFN-γ) gene encoding region is amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of bovine spleen lymphocytes stimulated with Phytohemagglut inin (PHA). The products of RT-PCR are cloned into PMD18-T vector. Postive recombinant clones named bovIFN-γ are identified by restriction enzyme digestion and sequencing. The fragment of bovIFN-γ the PMD-18T vector is subcloned into pGEX-4T-1. The pGEX 4T-1/bovIFN-γ is transformed to E. coli DH 5α.