CAJ | 학술논문

以重组质粒pMD18-T/IL6和pMD18-T/OmpW为模板，分别扩增红笛鲷IL-6基因和哈维氏弧菌外膜蛋白OmpW 基因，运用 PCR 重叠延伸剪切技术，将 IL-6和 OmpW 基因融合，将融合基因定向克隆到原核表达载体pET-32a(+)，转化大肠杆菌BL21（DE3）感受态，经异丙基-β-D-硫代半乳糖苷（IPTG）诱导融合蛋白高效表达，融合蛋白分子质量约为66.6 ku。优化后表达条件为温度37℃，IPTG 浓度0.2 mmol · L-1，诱导时间5 h。用HisTrap? HP 亲和柱纯化重组蛋白，最佳咪唑洗脱浓度为400 mmol · L-1，纯化蛋白的质量浓度为480μg · mL-1。Western-blot分析显示，该融合蛋白可与鼠抗His-tag单克隆抗体发生特异反应，表明目的蛋白得以正确表达。
이중조질립pMD18-T/IL6화pMD18-T/OmpW위모판，분별확증홍적조IL-6기인화합유씨호균외막단백OmpW 기인，운용 PCR 중첩연신전절기술，장 IL-6화 OmpW 기인융합，장융합기인정향극륭도원핵표체재체pET-32a(+)，전화대장간균BL21（DE3）감수태，경이병기-β-D-류대반유당감（IPTG）유도융합단백고효표체，융합단백분자질량약위66.6 ku。우화후표체조건위온도37℃，IPTG 농도0.2 mmol · L-1，유도시간5 h。용HisTrap? HP 친화주순화중조단백，최가미서세탈농도위400 mmol · L-1，순화단백적질량농도위480μg · mL-1。Western-blot분석현시，해융합단백가여서항His-tag단극륭항체발생특이반응，표명목적단백득이정학표체。
The fusion gene IL6-(Gly4Ser)3-OmpW was constructed with the DNA fragments of the red snapper IL-6 gene and Vibrio harveyi outer membrane protein OmpW gene which from recombinant plasmid pMD18-T/IL6 and pMD18-T/OmpW by Gene-SOEing method. The full length product was then cloned into the prokaryotic expression vector pET-32a(+) for protein expression in Escherichia coli strain BL21(DE3). The molecular weight of expression fusion protein IL6-(Gly4Ser)3-OmpW was about 66.6 ku. The recombinant protein was high expressed under induction conditions of exposure at 37℃, in 0.2 mmol·L-1 of IPTG for 5 h. The fusion protein was purified using HisTrap?HP affinity column and the best elution concentration of imidazole was 400 mmol·L-1. The concentration of purified fusion protein was 480 μg·mL-1. Western-blot analysis showed that the recombinant fusion protein could be combined with mouse anti-His-Tag Mab, indicating that the aim protein was expressed successfully. These results could provide a foundation for further study of its biological activity.