HBV pre-c-Fc融合蛋白在杆状病毒表达系统中的表达及其生物学活性研究

Expression and Purification of Biological-active Recombinant HBV Precore Protein-Mouse Ig G Fc Based on Baculovirus Expression Vector System

目的:在杆状病毒表达系统中表达融合蛋白乙型肝炎病毒前C蛋白-小鼠IgG Fc蛋白(HBV precore protein-mouse IgG Fc,HBV pre-c-Fc),并鉴定其免疫原性。方法:目的基因HBV prec-Fc连接到pFastBac1载体,获得的pFastBac1-HBV pre-c-Fc质粒转化DH10Bac感受态,通过Tn7转座子将目的基因转座到Bacmid中,得到Bacmid-HBV pre-c-Fc穿梭载体,脂质体包被后转染Sf9昆虫细胞获得P1代病毒,重复转染Sf9获得高滴度病毒。收集细胞上清超滤后通过Protein G亲和层析柱纯化得到目的蛋白HBV pre-c-Fc。纯化的蛋白大腿内侧肌肉注射免疫BALB/c小鼠并检测血清中乙型肝炎病毒核心蛋白抗体产生量。结果:HBV pre-c-Fc在昆虫细胞中成功表达,纯化后蛋白纯度达90%以上,蛋白产量约为3.03mg/L,纯化蛋白能有效刺激BALB/c小鼠产生特异抗体。结论:成功地在杆状病毒表达系统中表达了具有免疫原性的HBV pre-c-Fc蛋白,为生产乙肝治疗性疫苗奠定了基础。

Objective： The fusion protein HBV pre-C protein - mouse IgG Fc （HBV pre-c-Fc） protein was expressed based on the baculovirus expression vector system, and identify its immunogenicity. Methods： target gene HBV pre-c-Fc connected to pFastBac1 vector, pFastBac1-HBV pre-c-Fc plasmid was transformed DH10Bac competent, transposase Bacmid through Th7 transposon into the target gene, got Bacmid-HBV pre -c-Fc shuttle vector, Cellfectin ⅡReagent transfected Sf9 insect cells to obtain P1 generation virus, transfected Sf9 repeated to obtain high titer virus. Cell supernatants were collected after ultrafiltration to obtain the target protein HBV pre-c-Fc purified by Protein G affinity chromatography. The purified protein thigh intramuscularly immunized BALB/c mice and detected the concentration of serum hepatitis B virus core protein antibody. Results： HBV pre-c-Fc successfully expressed in insect cells, the purity of purified protein was over 90%, the production of protein is about 3.03mg/L, purified protein can effectively immunize the BALB/c mice to produce specific antibodies. Conclusion： The expression of immunogenic HBV pre-c-Fc protein based on the baculovirus expression vector system can be the production of hepatitis B therapeutic vaccine foundation.