CAJ | 학술논문

目的设计原核表达抗人白细胞介素-1β单链抗体(IL-1βscfv)与TNF-α可溶性受体的融合蛋白,并分析其生物学活性,为类风湿关节炎的治疗提供新的候选药物.方法利用RT-PCR方法从HeLa细胞总RNA中扩增了人肿瘤坏死因子Ⅰ型受体胞外区基因片段,与抗人IL-1βscfv通过抗体铰链区融合,克隆至pET27b(+)表达载体中,构建成pET-IL-1scfv:TNFR1质粒；转化大肠杆菌Rosetta进行表达.从包涵体中纯化得到IL-1scfv:TNFR1蛋白,进行IL-1scfv:TNFR1的鉴定及活性检测.结果 ELISA结果证明,IL-1scfv:TNFR1可以分别结合hIL-1β和hTNF-α,并呈现剂量依赖性,表明IL-1scfv:TNFR1的单链抗体部分和可溶性受体部分可以各自形成正确的空间构象；免疫斑点分析证明,IL-1scfv:TNFR1与hTNF-α结合后仍可以结合hIL-1β,具有同时结合hIL-1β和hTNF-α的两种靶分子的能力,表明IL-1scf:TNFR1的两个部分不会相互影响与靶分子的结合.活性测定结果表明,IL-1 scfv:TNFR1可以有效封闭肿瘤坏死因子对L929细胞的细胞毒效应,具有显著抑制hIL-1β促进L929细胞增殖的活性,IL-1scfv:TNFR1对两种靶分子的拮抗作用均呈现明显的剂量依赖性.结论成功构建了能够同时结合hIL-1β和hTNF-α两种靶因子的双特异性IL-1 scfv:TNFR1融合蛋白,并能有效抑制hTNF-α和IL-1β的生物活性,为类风湿关节炎的治疗提供新的候选药物.
목적 설계원핵표체항인백세포개소-1β단련항체(IL-1βscfv)여TNF-α가용성수체적융합단백,병분석기생물학활성,위류풍습관절염적치료제공신적후선약물.방법 이용RT-PCR방법종HeLa세포총RNA중확증료인종류배사인자Ⅰ형수체포외구기인편단,여항인IL-1βscfv통과항체교련구융합,극륭지pET27b(+)표체재체중,구건성pET-IL-1scfv:TNFR1질립；전화대장간균Rosetta진행표체.종포함체중순화득도IL-1scfv:TNFR1단백,진행IL-1scfv:TNFR1적감정급활성검측.결과 ELISA결과증명,IL-1scfv:TNFR1가이분별결합hIL-1β화hTNF-α,병정현제량의뢰성,표명IL-1scfv:TNFR1적단련항체부분화가용성수체부분가이각자형성정학적공간구상；면역반점분석증명,IL-1scfv:TNFR1여hTNF-α결합후잉가이결합hIL-1β,구유동시결합hIL-1β화hTNF-α적량충파분자적능력,표명IL-1scf:TNFR1적량개부분불회상호영향여파분자적결합.활성측정결과표명,IL-1 scfv:TNFR1가이유효봉폐종류배사인자대L929세포적세포독효응,구유현저억제hIL-1β촉진L929세포증식적활성,IL-1scfv:TNFR1대량충파분자적길항작용균정현명현적제량의뢰성.결론 성공구건료능구동시결합hIL-1β화hTNF-α량충파인자적쌍특이성IL-1 scfv:TNFR1융합단백,병능유효억제hTNF-α화IL-1β적생물활성,위류풍습관절염적치료제공신적후선약물.
Objective To express the anti-IL-1βscfv and soluble TNF receptor 1 (sTNFR1),and analyze their bio-activities.Methods sTNFR1 was obtained by RT-PCR from the total RNA of HeLa cells,and fused with IL-1βscfv by the hinge fragment of IgG molecule.The fusion gene IL-1scfv:TNFR1 was cloned into the expression vector pET27b(+).The fusion protein was expressed and purified from inclusion bodies.Results The ELISA analysis showed that the fusion protein could bind hIL-1β and hTNF-α respectively in a dose-dependent manner,indicating that scfv and sTNFR in the fusion protein can form the correct spatial configuration.The dolt-blot analysis showed that the fusion protein could concurrently bind with hIL-1β and hTNF-α,indicating that the combination of the two parts of the fusion protein does not influence each other for binding to their target molecules.The bioactivity assay showed that the fusion protein could inhibit both the cytotoxicity of hTNF-α on L929 cells and hIL-1β-induced proliferation of L929 cells,indicating that the fusion protein has the ability to neutralize hTNF-α and hIL-1β.Conclusion A bispecific fusion protein IL-1scfv:TNFR1 was successfully constructed.The fusion protein has the ability to inhibit the biological activity of hTNF-α and hIL-1β,and provides a drug candidate for the treatment of rheumatoid arthritis.