| Cholera, caused by Vibrio cholerae, is a acute and violent communicable diarrheal disease with characteristics of spreading quickly and severe damage. It is the first class of quarantined diseases in China and one of world seven pandemic deseases. There had been seven extensive epidemics in the world since 1817.So the prevention and control had been regarded by the government in many countries. Effective prevention of cholera dissermination depends on safe water sources and improved sanitation in countries where cholera is endemic. Poor economic situations in these countries make the cost of building large-scale water treatment and sanitation systems prohibitive. Vaccination is the most effective measures to prevent Cholera. Previous injection vaccines had many defects in different degrees. Studies showed that the vaccines of enteric communicable disease such as Cholera were more efficient by administering immunogens orally rather than parenterally. So, it had became the hot spot of developing safe,convenient,inexpensive oral vaccine.The plan"plant oral vaccine" was advocated by the World Health Organization (WHO) with the great significance to the implementation the vaccine. Our hope was to use the transgenic carrot as an edible vaccine to stimulate mucosal immune system producing protective antibody. Carrot is a very important fruit in the world, rich in nutrition, easy to eat raw and avoiding the breakage of the effective antigen.Our study is to transform choler toxin B (CTB) into carrot. The vector, which contained CTB under control of double CaMV 35S promoter and NOS terminator, were introduced into Agrobacterium tumefaciens strain, transfer the hypocoty of carrot. The kanamycin-resistant T0 and T1 plants, 110 and 172 respectively.Positive colonies were screened by PCR amplification, the universal primer n1/n2 and p1/p2 ,the special primer c1/c2 were designed and we found the positive rate were 81.8%,72.7% and 34.5% in T0 plants and 88.6%,74.3%,31.4% in corresponding T1 plants.The obtained transgenic plants possessed the good genetic stability.The protein level of CTB in the transformed carrot plants was 2.959μg pre gram root weight. The expression lever was up to 0.0423% per gram of total soluble carrot protein.The results indicated the CTB gene integrated in the carrot genomic DNA and the most were expressed, at the same time the levels were not very high overally. There were also no expression in some transgenic plants. The CTB specific expressions existed in some certain organs, such as leaves and roots and none in the stems.Western blot analysis indicated that carrot-derived CTB protein was antigenically indistinquishable from bacterial CTB protein,and that oligomeric CTB molecules(50 kDa) were the dominant molecular species isolated from transgenic carrot root tissue.Similar to bacterial CTB,plant-synthesized CTB dissociated into monomers(11.6 kDa)during heat treatment. It layed the foundation for providing some considerable base for further experimental of such edible vaccine.
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