Expression Of Cholera Toxin B Subunit And β-expansin Gene Specifically Expressed At Early Stage Of Wheat Male Gematophyte Development And Their Activities Assays In Vitro

1. Expression of cholera toxin B subunit in Bombyx mori baculovirus expression vector system and its activity assys in vitroThe non-toxic cholera toxin B subunit (CTB), with very strong immunogenicity, might help to stimulate mucosal IgA and serum IgG The principal receptor of B subunit is GMl-ganglioside, a glycosphingolipid found ubiquitously on the cell surface of karyotheca cells. It made B subunit to mediate receptor interactions that uptakes of its toxic A subunit. B subunit has more and more important role in study of immunoloical adjuvant, oral vaccine and vaccine derived from polypeptide.In this paper, the fusion gene encoding cholera toxin B subunit and part of polyhedrin was cloned into pBacPAK8, a baculovirus shuttle vector. The recombinant shuttle vector pBacPAK-mCTB was co-infected with Bm-BacPAK6 DNA into BmN cells. The recombinant virus BacPAK-mCTB was screened, plaque-purified and identified by PCR and Southern blotting.The BmN culture cells and the fifth instars larvae were infected by recombinant baculovirus BacPAK-mCTB. SDS-PAGE and Western blotting showed the anticipative bands corresponding to molecular weights of approximately 14kD and 70kD that were monomer and pentameric form. In ELISA assay, the expression product of cell and hemolymph of larvae were 5. 6μg/ml and 54. 4μg/ml, and GM1-ELISA showed high binding affinity of GMl-ganglioside.The immunogenicity of recombinant CTB was confirmed after NOD mice oral administration. It demonstrated that CTB could not only act as an effective mucosal adjuvant significantly increases the ability of CTB and insulin to induce immunological tolerance after oral administration tolerance but also as an immunogen enhances the induction of immunity. Titration of the CTB concentration in this system revealed that the ratio of insulin: CTB about 100μg: 10μg was optimal for the induction of oral tolerance. In addition, it significantly prevented the onset of diabetes in old NOD mice with established islet infiltration andincreased the specific tolerogenic effect of oral insulin.2. Clon of P-expansin gene {TaEXPBI) specifically expressed at early stage of wheat male gematophyte development and its expression in vitroUsing Suppression subtractive hybridization and RT-PCR, Northern, Southern technique, an microspore-specific beta-expansins cDNA clon (TaEXPB2 cDNA) was isolated from wheat microspore mRNA. TaEXPB2 cDNA transcripts are highly abundant in microspore and developing pollen at the early and middle stage, but undetectable in mature pollen, immature seed, larvae, stem, root and ovary. Southern hybridization and prediction of amino acid sequences indicated that TaEXPB2 cDNA might be a member of a small subfamily of expansins within a larger expansin family. It is significantly different from the group I allergen reported before which highly expressed in mature pollen.TaEXPBI cDNA was cloned into prokaryotic express vector pGEX-4T-l. The purified GST fuse protein showed that gene had been correctly inserted into the vector and to be induced express. A peptide about 20aa was designed and synthesized based on TaEXPB2 cDNA. It was used to immunise rabbit to gain polyclonal anti-TaEXPB2 antibody produced in rabbit. ELISA assay showed that the titer was at least 5X104 in immunoreaction. Then TaEXPB2 cDNA was also well expressed in BmNPV expression vector system for further bioactivity research in vitro. Now the identification of the recombinant virus BacPAK-TaEXPB2 is in the progress. The PCR identification supposed TaEXPB2 cDNA was well done in Baculovirus expression vector system. Therefore, it could be used to further expression research about its biological function.