CAJ | 학술논문

为了研究重组羊白细胞介素(gIL)-18在酵母系统中的高效表达,进一步阐明该重组蛋白的生物学活性,以含有gIL-18基因的重组质粒为模板进行PCR扩增,构建重组表达质粒pPICZ-gIL-18,转化于毕赤酵母GSll5,以甲醇诱导表达,经sDS-PAGE和western blot分析证实了重组蛋白的表达,分泌的重组gIL-18表达量为100 mg/L.经纯化后用MTT法和MDBK-VSV法检测表达的重组IL-18体外生物活性,利用免疫试验检测了重组蛋白对羊痘疫苗的免疫增强作用.实验结果表明,该蛋白具有诱导MDBK细胞分泌IFN-γ和刺激PBMC增殖的生物学活性,比活性为1.5×105 u/mg.体外具有增强羊痘疫苗的活性.毕赤酵母分泌表达的重组gIL-18具有良好的生物学活性,为gIL-18作为免疫佐剂和免疫治疗剂的大规模应用奠定了基础.
위료연구중조양백세포개소(gIL)-18재효모계통중적고효표체,진일보천명해중조단백적생물학활성,이함유gIL-18기인적중조질립위모판진행PCR확증,구건중조표체질립pPICZ-gIL-18,전화우필적효모GSll5,이갑순유도표체,경sDS-PAGE화western blot분석증실료중조단백적표체,분비적중조gIL-18표체량위100 mg/L.경순화후용MTT법화MDBK-VSV법검측표체적중조IL-18체외생물활성,이용면역시험검측료중조단백대양두역묘적면역증강작용.실험결과표명,해단백구유유도MDBK세포분비IFN-γ화자격PBMC증식적생물학활성,비활성위1.5×105 u/mg.체외구유증강양두역묘적활성.필적효모분비표체적중조gIL-18구유량호적생물학활성,위gIL-18작위면역좌제화면역치료제적대규모응용전정료기출.
For expression of goat interleukin-18 (gIL-18) in pichia pastoris system, the IL-18 gene was amplified by PCR and cloned into pPICZa A vectors, then transformed into pichia. pastoris system GS115. High level expression of recombinant protein up to 100 mg/L was achieved following inducing with methanol. Bioactivity of recombinant protein was analyzed by MTT and MDBK-VSV methods. The enhanced immunity was measured by goat pox vaccine test. The recombinant protein could stimulate PBMCs proliferation and induce IFN- γ production in MDBK cells. It also showed immune enhancement to the antigen of goat pox vaccine. The recombinant gIL-18 expressing in yeast system retained nature bioactivity and could be a valuable resource for the large-scale clinical use as an immune adjuvant or therapeutic cytokines.