奶牛γ干扰素基因的高效表达及活性测定

Expression and Activity Determination of Bovine Interferon-gamma Gene

经刀豆素(conA)刺激诱导奶牛外周血淋巴细胞,应用RT-PCR方法从其总RNA中对奶牛γ干扰素基因cDNA进行扩增,然后将特异性片段连接到pMD18-T载体,测序结果表明,与已知序列同源性为100%。然后将特异性片段连在pRLC载体上进行表达,经SDS-PAGE分析,原核表达产物为16kDa的重组蛋白,占菌体总蛋白的42%,表达产物以包涵体形式存在。经7mol/L盐酸胍的变性液溶解及0.5mol/L盐酸胍复性液处理,表达产物进行脱盐、凝胶层析纯化,细胞病变抑制法结果表明,重组牛IFN-γ具有较高的干扰素活性,约为6.0×105U/mg。

With the reverse transcription polymerase chain reaction (RT-PCR), the DNA sequence encoding the cattle’s interferon-gamma (BovIFN-γ) and the signal peptide was amplified, from the total RNA of the lymphocytes stimulated with concanavalin A in the peripheral blood of bovine, which was then cloned into vector pMD18-T and sequenced. The sequencing result showed that there is 100% homology among the documented sequences and sequence reported here, which was successfully inserted into the expressing plasmid pRLC and was highly expressed in E.coli. SDS-PAGE result showed that the cloned recombinant protein was expressed in the form of inclusion bodies in the E.coli cell with molecular weight of 16kDa and was amount to 42% of the whole protein in the E.coli cell, which was subsequently dissolved in 7mol/L guanidine chloride and renatured with dilution in refolding buffer containing 0.5mol/L guanidine chloride. In order to obtain pure protein, the renatured boIFN-γ was desalting by Hiprep 26/10 and purified by Hiprep Sephacryl S-200 chromatography. The purified product could inhibit the cytopathic effect, which verified it has the high cytokine activation.