CAJ | 학술논문

目的利用杆状病毒昆虫系统表达人干扰素(IFN) α-2b/免疫球蛋白(Ig)G4Fc融合蛋白(IFN/Fc),为长效干扰素的抗病毒治疗探索新方法.方法利用分子克隆技术获得人IFN α -2b及IgG4 Fc基因cDNA,构建杆状病毒穿梭载体,在DH10 Bac菌株中转座获重组杆粒,转染昆虫细胞High five,Western blot检测目的蛋白表达,细胞病变抑制法检测生物学活性. 结果穿梭载体在DH10 Bac中转座成功,获得重组杆粒Bacmid-IFN/Fc ; High Five细胞在病毒感染后72 h蛋白表达较好.病毒感染48 h即有少量目的蛋白表达,72 h表达量较高.Western blot结果显示在45×103处有特异性蛋白条带,体外抗病毒活性为580 IU/ml.结论利用杆状病毒昆虫系统成功表达重组IFN/Fc融合蛋白,为新型长效干扰素研制及慢性病毒性肝炎治疗提供实验依据.
목적 이용간상병독곤충계통표체인간우소(IFN) α-2b/면역구단백(Ig)G4Fc융합단백(IFN/Fc),위장효간우소적항병독치료탐색신방법.방법 이용분자극륭기술획득인IFN α -2b급IgG4 Fc기인cDNA,구건간상병독천사재체,재DH10 Bac균주중전좌획중조간립,전염곤충세포High five,Western blot검측목적단백표체,세포병변억제법검측생물학활성. 결과 천사재체재DH10 Bac중전좌성공,획득중조간립Bacmid-IFN/Fc ; High Five세포재병독감염후72 h단백표체교호.병독감염48 h즉유소량목적단백표체,72 h표체량교고.Western blot결과현시재45×103처유특이성단백조대,체외항병독활성위580 IU/ml.결론 이용간상병독곤충계통성공표체중조IFN/Fc융합단백,위신형장효간우소연제급만성병독성간염치료제공실험의거.
Objective To investigate a baculovirus insect cell system for expressing an interferon alpha 2b (IFNα2b)/immunoglobulin G-4 (IgG4) Fc fusion protein,which has long-acting antiviral effects.Methods Human IFNo2b and IgG4 Fc cDNAs were generated by molecular cloning and inserted into a baculovirus shuttle vector,which was then transposed into the DH 10 Bac strain to form recombinant Bacmid-IFN/Fc.The Bacmid-IFN/Fc was transfected into High five insect cells,and expression of the IFN/Fc fusion protein was detected by Western blotting and its biological activity was assessed by the cytopathic effect inhibition method.Results The IFNα2b and IgG4 Fc cDNA fragments were successfully amplified by RT-PCR using human peripheral lymphocytes.After cloning into the baculovirus shuttle vector,pFastBac 1,and transforming into DH10 Bac competent cells,screening identified positive clones carrying the recombinant Bacmid-IFN/ Fc.A Bacmid-IFN/Fc clone was successfully transfected into the High five insect cells and packaged into the baculovirus for expression of the IFN/Fc fusion protein.Western blotting revealed that the fusion protein expression was specific,and yielded a protein of 45 kD in size.The in vitro antiviral activity of the IFN/Fc fusion protein was 580 IU/mL.Conclusion A novel IFN/Fc fusion protein was successfully generated using a baculovirus insect cell system,which may prove useful for providing future experimental data for development of a new long-acting interferon to treat chronic viral hepatitis.