牛干扰素-γ基因的RT-PCR扩增、序列分析及其在大肠杆菌中的表达

Amplification sequencing of gene encoding the protein of bovine interferon-γ

根据GenBank上发表的牛干扰素γ基因序列设计引物,从牛外周血淋巴细胞中提取基因组RNA,采用RTPCR技术,扩增出干扰素γ基因并进行序列分析。琼脂糖凝胶电泳显示牛干扰素γ扩增片段约为500bp。序列分析结果表明,牛干扰素γ基因序列全长517bp,开放阅读框架内501个核苷酸,共编码166个氨基酸,分子质量19.4ku,与参考序列完全一致。克隆序列经BamHⅠ/EcoRⅠ双酶切后连接到经同样双酶切的PBV220质粒中,转化入大肠杆菌DH5α中,筛选阳性克隆进行温度诱导表达,经SDSPAGE分析,在约19.4ku处有表达的蛋白带,表达产物经免疫荧光染色鉴定为阳性。

Based on the published nucleotide sequence of bovine interferon - γ gene, a pair of RT - PCR primers were designed and synthesized. Totle RNA, isolated from peripheral blood T cells of bovine, was used as template to generate complementary DNA by reverse transcription. The 500 bp DNA fragment were amplified by polym- erase chain reaction. Sequencing results demonstrated that interferonygene contains 517 bp with a 501 bp open reading fragment （ORF） encoding a peptide of 166 amino acide whose molecular is 19.4 kd, which is completely accord with reference array. The cloning gene fragment was inserted into expression vector PBV220 when they are cut by BamHⅠ / EcoRⅠ. The recombinant plasmid was transformed into DH5α strain by CaCl2. The SDS - PAGE result showed that cloned recombinant protein was expressed in the supernatant with molecular weight of approximated 19.4Ku. The whole positire result has been certified by immune- fluorescence technique.