广西沼泽型水牛γ-干扰素基因的克隆与原核表达

Cloning and prokaryotic expression of Guangxi swamp type buffalo interferon-γ gene

本研究克隆了广西沼泽型水牛γ-干扰素(interferon-γ,IFN-γ)基因,并构建了水牛IFN-γ原核表达质粒。从健康沼泽型水牛静脉无菌采血,分离外周血单个核细胞(Peripheral blood mononuclear cells,PBMCs),用刀豆素A(Concanavalin A,ConA)诱导培养13 h后,提取细胞总RNA,用水牛IFN-γ基因特异性引物通过RT-PCR扩增水牛IFN-γ成熟肽编码区cDNA序列,将其克隆到pMD18-T载体中。RT-PCR产物电泳可见约457 bp大小目的片段。经过限制性酶切分析,测序证实克隆得到的基因序列正确。将IFN-γ成熟肽编码区基因片段切下亚克隆到表达载体pET-32a+中,构建成重组表达质粒pET-mIFN-γ。PCR、双酶切电泳和序列测定结果均证实已插入约457 bp的IFN-γ基因片段。经异丙基硫代-β-D-半乳糖苷(IPTG)诱导,水牛IFN-γ基因在大肠杆菌中获得了高效的表达,融合蛋白的分子量为35 ku,表达量占菌体总蛋白的43.6%。

Interferon-γ（IFN-γ） gene of Guangxi swamp type buffalo was clone and prokaryotic expressing plasmid containing buffalo IFN-γ gene was constructed. Total RNA was extracted from peripheral blood mononuclear cells （PBMCs） of healthy swamp type buffalo stimulated with Concanavalin A （ConA） for 13 hours. The cDNA of buffalo IFN-γ gene was amplified by RT-PCR using a pair of primers designed based on the published bovine IFN-γ gene sequence. The PCR products were cloned into vector pMD18-T. A 457 bp fragment of RT-PCR products was revealed by agrose gel electrophoresis. Sequence coding for mature buffalo IFN-γ protein was subcloned into an expression vector pET32a＋. Successful inserting of the 457 bp fragment into the vector pET32a＋ was confirmed by PCR, endonuclease digestion and sequencing. Positive recombinant clones were identified by restriction enzyme digestion and sequencing. The recombinant buffalo IFN-γ gene was highly expressed in Escherichia coli （43.6% of total bacterial proteins） after induced by Isopropylthio-β-D-galactoside （IPTG） and a remarkable 35 ku recombinant fusion protein was revealed by SDS-PAGE.