| Hepatitis B(HB) is one of the most widespread viral disease which is caused by hepatitis B virus. China is a high outbreak area where more than 10% of people suffering from this disease or carrying this virus. HBV can cause acute, chronic hepatitis and hepatocirrhosis even liver cancer. So far there is still none medicine to cure this disease and the uppermost measure to prevent the HBV infection is to inoculate HB vaccine. Hepatitis B surface antigen is the component of HBV envelope protein and has a favorable immunity quality. HBsAg used in vaccine was purified from the serum of infected individuals before a recombinant form (rHBsAg) was produced in yeast. The rHBsAg vaccine is the HBV gene engineering vaccine that is widely used. But its high produce cost, cold requirement during store and carry, and requiring specialties when inoculating restrict the utilization of this kind vaccine in many developing countries.At the beginning of 1990's, World Health Organization (WHO) brought forward want of vaccine which is cheap and do not require cold storage. Charles J. Antzen thought of a tentative plan, which is to produce edible vaccines by plant gene engineering. Namely the gene of the antigen can be transformed into plant and make it expressed in plant. Then individuals or animals, who take the plant or the antigen protein in plant, will produce immunity reaction of certain antigen. Up to now, the study of edible HB vaccine produced by transgenic plant has been proved to be possible and practical since HBsAg gene has been transformed into tobacco, potato, lupine callus, lettuce and peanut etc. But there is no report in strawberry yet.There are three advantages to produce edible vaccines using strawberry: 1) It changes the inoculation mode from injection to eating strawberry or drinking the juice; 2) It reduces the cost of vaccine production; 3) The transgenic strawberry plants can be grown in suitible areas, so the people in the bad medical condition can also be inculated by eating fresh strawberry. The safety production of HB vaccine simplifies the storageand delivery.The innovation of our study is to construct high expression vector of HBsAg in order to transform HBsAg to strawberry and produce HB edible vaccine using transgenic strawberry. This study will bring profundity meaning to prevent and control hepatitis B in our country.Our study focused on three aspects:1. A pair of primers was designed and synthesized based on HBsAg sequence registered in Genebank and an 810 bp fragment was amplified by polymerase chain reaction (PCR). The amplified fragment was inserted into pGEM-T vector. The sequence result showed that the target fragment was almost the same as that in Genebank.2. Plasmids SK(-) and pBin438 were used to construct a plant expression vector of HBsAg. pBin438 comprises of double CaMV 35s promoters, the ' &' fragment of TMV and a NOS-NPTII gene. The BADH gene was removed and the HBsAg gene was put under the control of double CaMV 35s promoters. The vector we constructed was named pBH.3. In order to testify the plant expression vector Agrobacterium mediated transformation was also performed in crowtoe using leaf disc. We obtained 36 kanmycin resistant plants and 9 of them were PCR positive. This proved that the vector could be used in transforming HBsAg gene into plant. This study constructed plant expression vector for strawberry transformation and established strawberry tissue culture system. This will facilliate the transformation of HBsAg gene into strawberry and the produce of edible HB vaccine. |
PDF Full Text Request