Studies On Expression And Transformation Of Human Hepatitis B Virus Large Surface Antigen Gene In Apple And Tomato

In this study, plant binary vector pYF9616 was constructed in which the PRS-S1S2S gene was controlled by tomato fruit-specific 2A11 promoter. Factors related to transformation of apple were studied. An efficient Agrobacterim-mediated regeneration and transformation system of 'Beni Aika' and 'Ryoka no Kisetsu' was developed. And we obtained the transgenic apple and tomato plants which can express and transcript normaly. The main results were as follow: 1. Construction of the plant binary vector:The fruit-specific promoter 2A11 was amplified from tomato genomic DNA using PCR techniques. The Hepatitis B Virus Large Surface Antigen Gene PRS-S1S2S Which contains tobacco PR-S signal in the N-terminal and SEKDEL ER retension signal at the C terminus was synthesized using PCR technique. Then the plant binary vector pYF9616 was constructed in which the PRS-S1S2S gene was controlled by fruit-specific 2A11 promoter.2.Establishing a high efficient regeneration and transformation system of apple leaf in vitro:The effects of different cytokinins, gelling agents, proliferative time, darkness, leaf polarity and concentrations of CH on regeneration of apple leaves were studied. And the efficient regeneration system was defined. On the MS medium containing TDZ 1mg/L and NAA 0.1mg/L, regeneration rate of Beni Aika apple leaf pieces whose addaxial surface touching medium can reached 100% after 15-20d dark culture .Then the concentration of TDZ 2mg/L and NAA 0.1mg/L was more properly for Ryoka no Kisetsu and we can get the regeneration rate of 95.2 %.Through Studying effects of Km selecting pressure concentration of Cb, Agrobacterium dipped time, co-cultivation time, the genetic transformation system had been established. On the basic medium MS containing TDZ Img/L and NAA0.1mg/L,the Beni Aika leaf was dipped in Agrobacteriwn suspension for 5min,then cocultivated with adhering Agrobacterim for 2 days in the dark. After 2 days on shooting medium with Km 12.5mg/L, Cb 250 mg/L. Then kanamycin-resistant shoots were obtained after 1-2 times selective cultivation. Ryoka no Kisetsu whose selecting medium contains Km6.25mg/L, Cb250 mg/L and basic MS medium containing TDZ2mg/L and NAA0.1mg/L.3. Distinguishing the transgenic apple , tomato, determining expression level of target protein and observing the rHBsAg from apple leaves and tomato fruit with electron micrograph of gold immunoaffinityThe PRS-S1S2S gene was introduced into apple cultivar Beni Aika by Agrobacteriwn tumefaciens-mediated transformation. The transgenic plants were obtained through kanamycin selection and GUS assay. The presence of the PRS-S1S2S gene was confirmed by PCR in apple and tomato. Moreover, the RT-PCR results showed that PRS-S1S2S gene has been expressed at the transcription level. In addition, ELISA results suggested the PRS-SJS2S gene was expressed correctly in transgenic apple plants and tomato fruit. The most amount of HBsAg in transgenic apple leaves is 11.180 ng/g fresh weight, and 523.53 ng/g fresh weight in transgenic tomato fruits. The expression of the transgenic PRS-S1S2S gene is very high in tomato fruit tissue, at least 110-285 times as high as the activity level measured in other organs coating the PRS-S1S2S gene drive by the 2A11 promoter. We observed that the rHBsAg from apple leaves and tomato fruits with electron micrograph of gold immunoaffinity. Inspection of those material revealed the presence of particles the average diameter was 22nm.We conclude that the rHBsAg made hi transgenic apple and tomato retain the capacity for self-association and thus has the physical properties of human serum-derived HBsAg and rHBsAg from yeast ,both of which are highly immunogenic in the particle form.