CAJ | 학술논문

目的:通过对葡萄球菌肠毒素 B(SEB)32位 His 进行定点突变,获得抑瘤效果显著增强的 SEB 突变体.方法:利用基因定点突变的方法,将 SEB 的32位 His 替换为 Asn,将重组质粒转入大肠杆菌中诱导表达,用 CM 弱阳离子层析柱纯化获得重组蛋白,用 SDS-PAGE 和 Western 印迹对其进行鉴定,并用增殖实验检测其丝裂原活性,通过 MTS法检测其体外抗肿瘤活性.结果:构建并高效表达了突变体蛋白 SEB(H32N),纯化获得了足够纯度的突变体蛋白;体外实验表明,在相同浓度下,SEB(H32N)的丝裂原活性及体外抗肿瘤活性明显优于野生型 SEB.结论:同野生型SEB 相比,突变体蛋白 SEB(H32N)对肿瘤生长的抑制作用得到了提高.
목적:통과대포도구균장독소 B(SEB)32위 His 진행정점돌변,획득억류효과현저증강적 SEB 돌변체.방법:이용기인정점돌변적방법,장 SEB 적32위 His 체환위 Asn,장중조질립전입대장간균중유도표체,용 CM 약양리자층석주순화획득중조단백,용 SDS-PAGE 화 Western 인적대기진행감정,병용증식실험검측기사렬원활성,통과 MTS법검측기체외항종류활성.결과:구건병고효표체료돌변체단백 SEB(H32N),순화획득료족구순도적돌변체단백;체외실험표명,재상동농도하,SEB(H32N)적사렬원활성급체외항종류활성명현우우야생형 SEB.결론:동야생형SEB 상비,돌변체단백 SEB(H32N)대종류생장적억제작용득도료제고.
Objective: The anti-tumor activity of staphylococcal enterotoxin B(SEB) was enhanced, by substitut?ing His32 of SEB for Asn through site-directed mutagenesis. Methods: A mutant superantigen SEB(H32N) was constructed by site-directed mutagenesis, the protein was expressed in E.coli BL21 after induced, purified by CM ion-exchange chromatography and identified by SDS-PAGE and Western blot. Mitogen biological activity was ana?lyzed by cell proliferation assay in vitro, and the anti-tumor activity was analyzed using MTS method in vitro. Re?sults: The mutated protein SEB(H32N) was expressed in E.coli efficiently and purified. The results showed that at the same concentration, the mitogen biological activity and anti-tumor effect of SEB(H32N) were better than SEB on a variety of tumor cells in vitro. Conclusion: Compared with the wild type SEB, SEB(H32N) mutant ex?hibited an enhanced anti-tumor effect.