乙肝病毒表面抗原基因在花生中的转化及免疫原性研究

【作者】 陈红岩；

【导师】 郑文竹； 夏宁邵；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2001， 硕士

【摘要】 花生(Arachis hypoaea)具有较高的营养价值，是一种廉价的植物蛋白质源。花生中含有约30％的蛋白质，因此是生产口服基因工程疫苗的理想载钵之一。本研究通过乙肝病毒表面抗原(HBsAg)在花生中的遗传转化研究，初步建立了花生的转化体系，并进行了初步的小鼠实验，结果表明花生表达的外源蛋白有良好的免疫原性，初步证实了转基因花生口服疫苗的可行性，为实现利用转基因花生生产大量、廉价的蛋白质疫苗提供一些实验和理论指导。受体系统：比较“泉花一号”、“汕油”、“鄂花四号”、“7868”四个花生栽培品种的半胚、小叶、胚轴等不同外植体的发芽率和发芽数，结果表明半胚最优，其次为胚轴和小叶。四个品种中又以“鄂花四号”的半胚发芽率和发芽数最高，可达92.5％和9-10个芽。 植物表达载体的构建：将乙肝表面抗原(HBsAg)基因插入植物双元表达载体pCAMBIA1301中，采用花椰菜花叶病毒(CaMV)35s启动子，Tnos为终止子，以gus基因为报告基因，植物抗性基因为hvg基因。 转化系统：对农杆菌转化条件的比较发现，菌株、共培养时间、共培养条件等均对转化有较显著的影响。对3个菌株(LBA4404、AGL1、EHA105)侵亲染力比较中发现，用EHA105浸染花生的转化效率最高。另在提高细胞感受态方面，在菌液和共培养基中加入0.2 mol/L CaCl(?)共培养时间由常规的2-3d增加至5d，可有效的提高转化效率。 转化体获得：选择筛选剂及确立基础抗性时，发现花生半胚对潮霉素比卡那霉素敏感，在潮霉素浓度为20mg/L时就可以完全抑制芽的分化。转化植株的筛选时，比较了间歇加压法和持续加压法。通过间歇式加压法从46个外植体中得到5株抗性体，但均未见报告基因(gus)表达，假阳性率高达100％；经持续式加压法从85个外植体中得到3株抗性体，其中2株有gus基因表达，因此最终选择持续加压法进行筛选。从花生“鄂花四号”成熟胚120颗中，利用半胚诱导、持续加压最终获得3个转化植株，均有明显gus基因表达。转化体外源基因槛、表达酌检测：对获得的转化植株，经PCR、peR—Sou山ern杂交和点杂交，证实目的基因己整合到花生基因组中，ELISA检测表明花生中表达的乙型肝炎表面抗原具有良好的活性，并初步定量计算出花生小芽中HBSAg蛋白含量分别约为1．27 X 10；g／g、2．4X 10”g／g和1．21X10rtg／g潍。免疫原性检测：取HBSAg ＆jkgf高的植株巳的叶片经诱导愈伤，高压筛选扩繁出大量愈伤组织，分别以蛋白粗提液和部分纯化的重组蛋白，肌肉注射初兔一次的小鼠，抗体均有明显的上升趋势，表明转基因植株中表达的重组HBSAg具有良好的免疫原性。取同样的转化硼组织，敏饲喂己免驭懒体下降至0．025（HBsAb ELISA皿值）的Balb／c小鼠，在第 5周出现抗体回升，第7周抗体ODM至3．54，而饲喂未经初兔的小鼠，直至第7周仍未见明显抗体产生。这些结果表明转基因植株经口服可以诱导回亿性抗体产生，而目前的表臼主量尚不足以作为初兔疫苗。 本研究首次将花生半胚作为转化受体用于花生遗传转化，并表明了其相对于常规胚轴和子叶具有一定的优势，在此基础上优化了农杆菌介导的花生半胚的遗传转化系统，并首次实现了HBSAg基因在花生中的表达，初步证实了转基因花生作为口服疫苗的可行性，为今后研制具实用价值的转基因花生口服疫苗奠定了坚实的基础。更多还原

【Abstract】 AbstractPeanut has high nutritious valtE, ami also is a [ow-cost source of protein Tbis about 30 % protein in peanut .Thus it is one of tIE ideal vector in oral vaccine of transgenc plant In this study, through research of transforming HBsAg into peanut, x~e ha~ e set up a transformed system of peanut, and limited experiments with mice,i,uw thu ii icwmbit~Mit iiBsAg from traiisfuuned peanut has good iinniunogenecity These results indicated the feasibility of transgenic peanut as oral vaccine, and provided some indexes for the production of large-scale, low-cost vaccine using transgernc peanutAcceptor system Rate of caespitoso bud production and number of regenerate bud were compared among different explants(half-embryo,lillter leaf and radical) and different breeds(’~uanhua No I ","Shanyou’Ve hua No 4" and "7868"). The results showed that half-embryo is better than the little leaf and the radicel, and the half-embtyo of breed "e [ma No 4" is best, whose caespitoso bud rate is as high as93.5%, and can get 9 to 10 regenerated buds per explant.Construction of plant exprcs~ed vector Hepatitis B surfiice antigen (I-IBsAg) gene was inserted into plant expressing plasmid pCAMBIA 1301 under the control of the CaMV 35s promoter and nos terminator, gus gene is also included in this vector as reporter gene, and the selectable maker gene for the vector is neomycin phosphotransferase geneTransformed system Strains of Agrubacterium tumelàciens, time of co-culture and condition of co-culture can make great eflèct on transformation Among three Agrobacteiium tiunefàciens strains, LBA4O4, AGL and EHA1OS, the half-embryo is the most sensible to EL-LA 105 It is an adaptable pathway to prolong the co-culture time from two days to five days and addition of 0.2M CaCI2Obtained and detection of transformed plant Result of basic 1~stness of half embryo to Kan and Hyg show that the Hyg was more efficient for selecting transformants than Kanawiia Only with 20 mg’L of Hyg, the growlh of bud would be restrained Two screen means, the discontinuous stress selection and continuous stress selection, e compared From 46 explants, five buds ~vete survived under discontinwus stress of Hyg, but the reporter marker (gus) could not be detected fitwn all of the2m In comparison, from 85 explants, three buds were survived undercontinuous stress of Hyg, and the reporter marker (gus) could be detected from twoof them, so the continuous stress method was chosen to screen transformed plant From 120 half-embryo of "e’hua No 4" peanut, three transfonned plants were obtained at last after co-culture with Agrobacterium tumefaciences EHA1O5 (including the pl3OlHBs) and hygromycin selection.Foreign gene integrated and express analysis of transformed plant The presence and integration of foreign DNA in transgenic peanut was confirmed by hygromycin resistance, GUS detection, polymerase chain reaction (PCR), PCR-southem blot and genomic dot blot analysis. The immunoactivity of recombinant NBsAg (rHBsAg) was shown by ELISA. The amount of rHBsAg in transgenicbudofpeanutisaboutl.27X1(Y’g ~. 2. 4X1(17g and 1. 21X1(Y7gperg fresh weight respectively.Immunogenicity of ILBsAg A great deal of regenerated callas were obtained fmm leaf of a transgenic plant Inimunogenicity of rHBsAg derived from lransgenic peanut callus was confirmed by muscle injection of raw protein extraction and purified protein extraction from transformed callus, and oral feed of transformed callus to pre-vaccine Balb/c mice with commercial HBV vaccine. Special antibody to HBsAg were boosted in all the cases.In our study, half-embryo was used as the acceptor of peanut transformation for the first time. The results proved superiority of half- embryo compared with radicel and little leaf After optimizing the transformative procedures, the HBsAg was transformed into peanut and the expression and immunoactivity of the rHBsAg were also confirmed The feasible of peanut as oral vaccine is also proved, which provided a basic confidence更多还原