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砚委
7 years ago
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4 Abstract Hepatitis B virus infection is one of the most widespread viral infections of humans. China belongs to high epidemic area. Chinese government has listed HBV immune into planned immune system since Every newborn needs to accept HBV immune. And it is a development tendence in many areas of the developing world which belong to high epidemic areas of HBV, too. It is about 10 years since the first group of newborns accepted HBV planned immune. Serum antibody titers of most of them have descended to a lower level. They need to accept boost immune to maintain the antibody s protection function from viral infection. The existing intramuscular injection vaccine of rhbsag results in effective immunization and protection in infants. For boost immune, however, because parenteral delivery needs to be administrated in special conditions and will cause some pain, it isn t easy to be accepted widely. In recent years, a novel production system of vaccines edible vaccines or oral vaccines is being developed. Although the licensed HBV vaccines developed to date have been for parenteral delivery, there is no a prior reason to exclude oral delivery. Oral vaccines can serve multiple immunization priorities, including simplicity of use, increase in compliance (as a result of increased ease/comfort of delivery), enhanced immune responses at mucosal sites, and stimulation of humoral immunity. Cherrytomato is a kind of novel fruit vegetable. It has high nutritious value and can be consumed raw. And the transformation system of tomato is easy. Thus cherrytomato is one of the ideal species for expressing subunit vaccine components. The hepatitis-b surface antigen (HBsAg) gene linked with CaMV35S promoter was respectively inserted into mini Ti plasmids of the binary expression vector pcambia1301 (containing hygromycin-resistant gene, kanamycin-resistant gene, Gus gene) and pcambia1300 (containing Hyg r gene, Kan r gene, no Gus gene), to form the reconstructed plant expression plasmids (p1301hbs, p1300hbs). In the same way, the plant expression plasmids (p1301ne2, p1300ne2) carrying hepatitis-e virus open reading frame-2 (HEV-NE2) gene were constructed. Tested by restriction endonuclease analysis, these plasmids were directly introduced into three strains of Agrobacterium tumefaciens (LBA4404, EHA105, AGL1). Sensitiveness to Agrobacterium tumefaciens and rate of caespitosa buds production were compared between the cotyledons and hypocotyls of breed purple husk. The results showed that cotyledon was the better acceptor material and the buds coming from cotyledon have better quality. 4

5 Strains of Agrobacterium tumefaciens, time of co-culture and condition of co-culture can make some effect on efficiency of transformation. Among three strains of Agrobacterium tumefaciens, the cotyledon of cherrytomato is most sensitive to EHA105. And the suitable co-culture time is 3 days. After the procedure of killing Agrobacterium tumefaciens, the explants should be resumed for about 1 week, and then transferred to culture medium containing antibiotic. Resuming culture will do good to efficiency of transformation. Mediated by EHA105, the mini Ti plasmids containing HBsAg and HEV-NE2 were transferred to cherrytomato. After 3-4months, many hyg-resistent plantlets were acquired. By far, we have got 10 stains of cherrytomato containing p1301hbs, 8 strains of cherrytomato containing p1301e2, and the hyg-resistent calli containging p1300hbs and p1300e2 respectively. The presence and integration of target gene in transgenic cherrytomato was confirmed by hygromycin resistance, histochemical detection of Gus activity, polymerase chain reaction (PCR) and Southern dot bloting analysis. The immunological activity of recombinant HBsAg, HEV-NE2 was shown by ELISA. The expression of rhbsag is better. The expression levels of rhbsag derived from defferent organs of the same plant have been compared, and we found the expression level of rhbsag in cherrytomato s leaf was highest. It can get to 300ng/g fresh weight. And the rhbsag expression level in fruits was about 10ng/g fresh weight. In this study, the possibility the inequality of the expression levels between Gus gene and target gene has been discussed primarily, too. Immunogenicity of rhbsag derived from transgenic cherrytomato was confirmed by immunization of mice. Prime and boost in muscle injection with purified plant protein extracts extracted from transformed tissues and direct oral feeding with transformed tissues have been done respectively. The results showed that the effectiveness of boost is better than prime. After the serum antibody level of the mice administered parenteral prime with 2µg commercial HBsAg vaccine began to descend apparently, three of them began to be fed transgenic cherrytomato tissues (about 1µg HBsAg every day) and two was injected purified transgenic plant protein extracts (about 0.6µg HBsAg). After 2-3 weeks, all of these mice elicited strong secondary antibody response. The result of this animal immune test indicated the rhbsag derived from transgenic cherrytomato has normal immunogenicity. Through Electron micrograph, obvious round particles can be found in the purified transgenic plant protein extracts, and it is similar to HBsAg in nature. 5

6 By far, we have got the second generation of cherrytomato transformed p1301hbs, and strong Gus activity has be detected in its leaf. In this study, we choose a new foreign breed of cherrytomato purple husk. Through the research of transforming HBsAg gene and HEV-NE2 into cherrytomato, we have set up a high efficient transformation system of cherrytomato, and the recombinant protein from transgenic plants has been proved to have good immunogenicity. The results of this study indicated the feasibility of the production of oral vaccines by transgenic cherrytomato, and provided some theoretic and experimental directions for the production of large-scale, low-cost oral vaccines using transgenic cherrytomato. Key words: transgenic plant, oral vaccines, cherrytomato, hepatitis B surface antigen (HBsAg), hepatitis E virus (HEV). 6

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17 Degree papers are in the Xiamen University Electronic Theses and Dissertations Database. Full texts are available in the following ways: 1. If your library is a CALIS member libraries, please log on and submit requests online, or consult the interlibrary loan department in your library. 2. For users of non-calis member libraries, please mail to etd@xmu.edu.cn for delivery details.

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