Analysis of the limitations of hepatitis B surface antigen expression in soybean cell suspension cultures
| Autor: | L. Srinivas, G. B. Sunil Kumar, Vishwas A. Bapat, Thumballi R. Ganapathi, C. J. Revathi |
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| Rok vydání: | 2007 |
| Předmět: |
DNA
Bacterial HBsAg Time Factors DNA Plant Blotting Western Cell Culture Techniques Enzyme-Linked Immunosorbent Assay Plant Science Microbiology Transformation Genetic Plant Growth Regulators Western blot Gene expression medicine Sorbitol Mannitol Hepatitis B Surface Antigens biology medicine.diagnostic_test Reverse Transcriptase Polymerase Chain Reaction Cell growth Antibodies Monoclonal General Medicine Agrobacterium tumefaciens Plants Genetically Modified biology.organism_classification Molecular biology Culture Media Blot Cell culture Azacitidine biology.protein Soybeans Antibody Agronomy and Crop Science Genome Plant |
| Zdroj: | Plant Cell Reports. 26:1575-1584 |
| ISSN: | 1432-203X 0721-7714 |
| DOI: | 10.1007/s00299-007-0379-7 |
| Popis: | Soybean cell suspension cultures were transformed using Agrobacterium tumefaciens harboring pHBS/pHER constructs to express hepatitis B surface antigen (HBsAg). The transformed colonies were selected and analyzed for the expression of HBsAg by PCR, reverse transcription (RT) PCR, Western blot and ELISA analysis. The maximum expression of 700 ng/g F.W. was noted in pHER transformed cells. The highest expressing colonies were used to initiate the cell suspension cultures and the expression of HBsAg was estimated periodically. The expression levels were reduced drastically in cell suspension cultures compared to the colonies maintained on semi-solid medium. Various parameters were studied to maximize the cell growth and to retain the expression levels. The supplementation of culture medium with a protease inhibitor, leupeptin hemisulfate could restore up to 50% of HBsAg expression in cell suspension cultures. This is the first report to investigate the possible cause and solution to the loss of recombinant protein expression levels in plant cell suspension cultures. |
| Databáze: | OpenAIRE |
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