| Hepatits B virus infection is one of the most widespread viral infections of humans, especially in China. A highly effective and safe hepatitis B vaccine was licensed in the U.S. in 1986. It has a great commercial success, with sales in the industrialized world exceeding $ 1 billion every year. Why have we pursued to an alternative vaccine against hepatitis B virus (HBV) infections? The answer lies in finding an equivalent or improved vaccine to serve for developing countries of the world, where the HBV disease burden remains at epidemic levels, and the price of vaccine has precluded its popularity.The existing vaccine against HBV infection is a biotechnological product that falls in the category of "subunit vaccines". The gene coding the hepatitis B surface antigen (HBsAg) was expressed in yeast or CHO cells. The resulting vaccine is the epitome of modern macromolecular pharmaceuticals derived from recombinant DNA technology; it is a superior product that contains only a "subunit" of HBV. However, it is technology-intensive and expensive that the vaccine could not be a health-care product for developing countries.In this paper, immunogenicities of recombinant hepatitis B surface antigens derived from CHO cells and in transgenic ginseng cells were compared. Transgenic cells material containing HBsAg was propitious to induce an immune response. HBsAg-S gene was obtained from C28 cell containing the HBsAg-S DNA fragment using PCR and inserted into vector pMD18-T, to form pMDBs. The recombinant plasmid pMDBS was digested with XhoI and reaction of filling the ends with T4 DNA polymerase was carried out, then degested again with SstI. The DNA fragment recovered by electrophoresis was ligated into the binary plant vector pBI121 digested by SmaI/SstI, the resulting plasmid, pBIBSa was gotten.The pBIBSa was transformed into Agrobacterium tumefaciens stain LBA4404 directly by the freeze-thaw method. Subsequently, Agrobacterium tumefaciens carrying pBIBSa was used to transform ginseng cells. Ginseng cells were co-cultivated for 48-72 h with Agrobacterium tumefaciens carrying pBIBSa. Ginseng cells were then rinsed with sterilized water added 500mg/L ampicillin to kill the Agrobacterium tumefaciens on the surface of cells, and blotted dry on a sterilized paper towel, and placed onto a 67-V medium added 500mg/L ampicillin for recovery. After recovery period of 7 days, the ginseng cells were transferred to a 67-V medium added 300mg/L ampicillin and 50mg/L G418 to select transgenic progeny. About 3-4 months later, G418-resistant ginseng cells regenerants were removed to a 67-V medium containing 35mg/L G418. We have obtained the cell line of ginseng, named CS83 line which carrying HBsAg-S gene. At the same time, CS83 cell suspension culture system was constructed.Transgenic cells were checked for the presence of target gene using PCR and RT-PCR. Samples containing the target gene showed a clear band in site of 700bp by agarose electrophoresis analysis. The results demonstrated that an amplified product with expected size was present in G418 resistant cells and absent in non-transformed cells.CS83 cells and negative control non-transformed ginseng cells were harvested, frozen in liquid N2, and ground to fine powder. The powder was suspended in extraction buffer (50mM Tris-HCl, 0.029% NaN3, PH9.5) at room temperature overnight. The surry was then centrifuged at 12000 rpm for 10 min at 4℃, and supernatants were tested using ELISA. Extracts of the CS83 line expressed HBsAg detected by ELISA. Expression levels of HBsAg showed 124ng/g fresh weight, and the HBsAg amount was 0.006% of the total soluble protein. The untransformed ginseng cells did not express any protein that was reactive to antibody against HBsAg. HBsAg was expressed in intracellular and secreted forms in suspension culture CS83 cells. Western blotting analysis confirmed the presence of 25 kDa band specific to HBsAg in CS83 cells. Forty-eight mice in six groups were injected. Group 1 were celiacly injected with 1 ml supernatant...
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