| Hepatitis B is a severe infectious disease caused by Hepatitis B virus (HBV) infection. About 10% of peoples are HBV carriers in China. Currently, there is no effective treatment for this disease, and prevention of it mainly depends on vaccine. But, commercial vaccine produced in yeast expression system is expensive for its higher production costs, and needs refrigeration. This made the universal vaccination difficult in remote areas and economically underdeveloped regions. In addition, about 10% of the population had no immunological response to the commercial vaccine. Thus, it's the exploration of a new form of HB vaccine that comes to people's attention. In sum up the work previously studied, the novel modified Hepatitis B surface antigen gene SS1 was expressed by an optimistic plant production system in this study, and some important results were discribed below.1, To detect wheher the the new hepatitis B antigen SS1 studied in this study, in which is the precursor peptide preS1 (21-47aa) was fused to the C-terminal of the hepatitis B major surface antigen (S), could arouse anti-preS1 antibody protection reaction in vivo through its immunization except for anti-S anitibodies, the full length preS1 gene (adr serotype) with codon preference in accordance with the E. coli strain B was synthesized and expressed in E.coli for preparetion for detection of anti-preS1 antibodies. The result showed that the recombinant proteins were in soluble form, accounting for about 44% of total protein. After purification with Ni-NTA affinity chromatography, the purity of purified protein reached to 98%. The result of Western blot and indirect ELISA showed that protein is stable and has preS1 antigenicity which was comparable to that of the commercial preS1. Results showed that we have overcome the problems encountered in the previous studies on full-length preS1 protein expression, such as products in the inclusive body form, protein unstable, or low expression. It provides the basis for the animals' clinical study of this new hepatitis B vaccine.2, For exploration of a new form of HB oral vaccine, the rice was used as an expression host of the recombinant protein attributing to that more and more studies showed that grain will be the optimistic expression system for producing the plant vaccine in the future. In addition, rice is the major food source for people in Asia, and is a self-pollinated crop that would relieve the transgene contamination caused by target gene flowing. In this study, we selected promoter of GluB-4 gene with higher activity to drive the foreign gene SS1 according to the reported papers and constructed a rice seeds-specific expression vector p1300GSS1 for transformation. Through transformation by Agrobacterium-media, the SS1 gene was inserted into the rice genome, and the highest expression level of the recombinant protein was about 31.5 ng/g DW seeds. It could self-assemble into virus-like particles structure with diameter of about 22±2 nm and a sedimentation coefficient of 1.25 g.cm-3. Western blot analysis revealed that the recombinant protein have both S and preS1 antigenicity. Animal experiments showed that recombinant protein in mice could provoke antibody against S and preS1. These results will encourage us to further evaluate the oral immunogenicity of this recombinant protein in the subsequent clinical study.3, In order to further improve the expression level of the recombinant protein SS1 in plant, the chloroplast expression system was tried firstly. An increasing number of studies show that plant chloroplast expression system has many advantages, of which high expression level of foreign proteins was most striking (the highest expression achieved to 46.7%TSP). Therefore, chloroplast expression system used for producing hepatitis B surface major antigen was another strategy to enhance the expression level. However, there is no application of this expression system for the expression of hepatitis B major antigen to date. Many review literature showed that the protein expressed in the chloroplast could not be glycosylated, but there were no specific studies related to the reports to prove it. Hepatitis B surface antigen is a glycoprotein, and its glycosylation-degree directly relates to its antigenicity. In this study, a tobacco chloroplast expression vector was constructed. As a model plant, tobacco was firstly used for tentative expression of hepatitis B surface antigen. The result showed that foreign gene was correctly integrated into the predetermined site of the chloroplast genome. After four rounds of selection, the transgenic plants were not homoplastic yet. The recombinant protein was detected to be expressed in the transgenic plant by hepatitis B surface antigen detection kit, but the expression level was very low. This may relates to the unhomoplastic and unglycosylation. Related research is still continuing.4, To achieve a good immunological effection, an immune adjuvant protein CTB (cholera toxin B subunit) was also expressed in E.coli. in this study. The CTB gene encoding the cholera toxin B subunit without the signal peptide-coding sequence was cloned, and constructed into the E. coli expression vector pETCTB for its expression. The result showed that the recombinant protein was expressed in the form of inclusion bodies, accounting for about 25% of the total protein. Through the denaturation and refolding of inclusion bodies, the purity of recombinant protein CTB reached to 98%. The result of bioactivity detection in vitro showed that the purified recombinant protein was mainly presented in form of pentamer. The result of western blot showed that the protein in form of pentamer were about 66.8% of total purified protein.This established expression system of oral adjuvant will help the further oral experiments of this recombinant protein.
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