| 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... |
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