The Cloning And The Expression Of HBsAg Gene In Pichia.pasteoris And Grape Tissues

This research amplified HBsAg gene (adr) by PCR,we constructed the yeast and plant expressive vectors.The expression of HBsAg in Pichia.pasteoris and grape tissues were studied. The main achievements were as follow:1 HBsAg gene was amplified from the pBI121/HB,then HBsAg gene was cloned into pUC19.The recombinants were assayed by the digestion of restricted enzyme and sequenced.The sequences were blasted in the NCBI.The results of sequences were consistent with the sequence of AY646444.1.The results indicated that the HBsAg gene was cloned into pUC19.The results built a solid base for the construction of expressive vector.2 The clone vector and pPIC3.5K were digested by the restricted enzyme,then ligated by T4 DNA ligase.The HBsAg gene was cloned into pPIC3.5K by analysis.The expressive vector was transformed into Pichia.pasteoris (GS115) by LiCL method. Transformants were obtained by PCR.The recombinants were obtained by the assay of PCR. Then the strain express the HBsAg in the culture medium using methyl alcoholas the carbolic rescource.The expressed protein was the most at 72 hr by SDS-PAGE.Expressed protein were 23kDa and 27kDa,this indicated protein were modified by GS115.ELISA assay were positive,it showed that the expressed proteins were immunocompetence.So the HBsAg gene can be use for expression research.These results assured the transformation of plant.3 In this work,the new plant expressive vector(pCAMBIA1301/HB) was constructed successfully.The pCAMBIA1301/HB was transformed into agrobacterium LBA4404 by freeze and thaw method.Then the grape calli was transformed by LBA4404.4 In this experiment, the calli of Vitis vinifera was material that wasinduced from leaves for transformation. The grape calli were induced in MS with 1mg/L NAA , 0.1mg/L6-BA NAA.And we made a pre-experiment to culture the grape calli with different hygromycin B concentration. The results indicated that the hygromycin B could kill the cell of grape calli at 30 mg/L completely. Hygromycin B can inhabit the growth of grape calli at 20 mg/L,so the selective concentration of hygromycin B was 20 mg/L.5 The grape calli were precultured and pre-treated by mannitol before transformation ,the results indicated that the transformation frequency were the highest at 3 days .The transformation frequency reached to 50%.The grape calli were treated with manitol at 7 concentration(0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7mol/L) respectively.The transformation results indicated that the transformation frequency was the highest when manmitol concentration was 0.4mol/L and culture time was 4 hr,the treatment increased the transformation frequency of agrobacterium evidently,the transformation frequency reached to 60%.6 The HBsAg gene was transformed into grape calli by assay of PCR and southern.A expressive system of transgenic grape tissues were constructed.The transgenic grape calli were obtained successfully.