乙肝病毒表面抗原基因在花生中的遗传转化及免疫原性检测

Transforming HBsAg into peanut and detection of its immunogenicity

首次用花生半胚作为外植体,与农杆菌EHA105(含质粒p1301HBs)共培养5d,再生培养基中通过加入潮霉素进行抗性筛选,得到抗潮霉素的芽,经芽的伸长、诱导生根获得转化植株。经PCR、PCR-Southern杂交、Southern点杂交等分子检测,证实目的基因已整合到花生基因组中,ELISA检测证实了在花生中表达的HBsAg具有较好的活性。经初步定量,花生小芽的蛋白初提液中可溶性蛋白含量1.044g/L,HBsAg小蛋白的含量约占总可溶性蛋白的0.032%,每克转化植株小芽鲜重含HBsAg小蛋白约2.4×10-7g。转基因花生植株初提重组蛋白经HPLC纯化、浓缩后,注射初免一次的小鼠,有明显的特异性抗体产生。口服饲喂已免疫但抗体下降至0.025(HBsAbELISAD值)Balb/c小鼠,发现有较强的抗体回升,达3.54,表明转基因花生疫苗可以加强口服免疫。

Half-embryo was used as the acceptor of peanut transformation for the first time.Half-embryo co-culture with Agrobacterium tumefaciens strain EHA105(including plasmid p1301HBsAg)for five days.Through hygromycin selection,transformed peanuts were obtained.The presence and integration of foreign DNA in transgenic peanut was confirmed by hygromycin resistance,GUS detection,polymerase chain reaction(PCR),PCR-southern blot and genomic dot analysis.The immunoactivity of recombinant HBsAg(rHBsAg)was shown by ELISA.The amount of rHBsAg in transgenic bud of peanut is about 2.4×10 -7 g /g.Immunogenicity of rHBsAg derived from transgenic peanut callus was confirmed by muscle injection of raw protein extraction and purified protein extraction from transformed callus,and oral feed of transformed calli to pre-vaccine Balb/c mice with commercial HBV vaccine.Special antibody to HBsAg were boosted in all cases.The feasible of peanut as oral vaccine is also proved,which proved a basic confi-dence for developing a practical transgenic peanut oral vaccine.