Expression Of Cholera Toxin B Subunit (CTB) And Its Fusion Protein With Polyhedrin (4aa-CTB) In Silkworm BmNPV Expression Vector System

The non-toxic cholera toxin B subunit(CTB),with very strong immunogenicity,might help to stimulate mucosal IgA and serum IgG.The primary function of B subunit is in mediating receptor interactions that uptakes of its toxic A subunit.The principal receptor of B subunit is GMl-ganglioside,a glycosphingolipid found ubiquitously on the cell surface of karyotheca cells.B subunit has more and more important role in study of immunoloical adjuvant,oral vaccine and vaccine derived from polypeptide.Baculovirus expression vector system (BEVS) is a high-level eukaryotic expression system founded in 1980's. Up to now thousands of genes have been high expressed by or using this system. Silkworm (Bombyx mori) BEVS has some additional advantages as compared to normal BEVS.In this paper, the cDNA encoding cholera Toxin B Subunit(CTB) & fusion protein of cholera toxin B subunit and polyhedrin (4aa-CTB)was cloned into pBacPAK8, a baculovirus shuttle vector. The recombinant shuttle vector pBacPAK-CTB and pBacPAK-4aa-CTB was co-infected with Bm-BacPAK6 DNA into BmN cells. The recombinant virus was screened, plaque-purified and identified by PCR and Southern hybridization.BmN culture cells were infected with the BmBac-CTB and BmBac-4aa-CTB at a MOI of 10. The cells were collected after 48 hours, then lysed with PBS for preparation of cell extract. SDS-PAGE analysis indicated that the LacZ gene has been replaced by CTB and 4aa-CTB gene in the recombinant viruses. In ELISA assay, the expression product of cell can show activity with P/N value of 3.6.The recombinant virus was also expressed in the larvae of silkworm by inoculation of recombinant virus into the 5th instar silkworm. Hemolymph of the infected larvaewas collected every day. The ELISA assay showed a maximum activity in day 6; the biological activity assay show that the expression product in larvae is about 10-fold higher than that expressed in cultured cells. As expected, SDS-PAGE and Western blotting analysis showed a pattern of molecular weight about 14.4kD.The expressed CTB and 4aa-CTB. was determined by ELISA after infecting Bm-N cells and larvae of silkworm with recombinant virus BmBac-CTB and BmBac-4aa-CTB.The results showed that expression amount reached 3μg/ml and 30μg/ml with BmBac-4aa-CTB in Bm-N cells and larvae of silkworm respectively.On contrary, expression amount reached 2.4μg/ml and 24μtg/ml with BmBac-CTB in Bm-N cells and larvae of silkworm respectively. Expression amount in the former was increased 20% and 30% respectively.