Generating Stable Cell Line for Producing Recombinant Phospholipase A2 of Honey Bee (Apis mellifera)

BACKGROUND:

 Honey bee venom contains complex compounds such as polypeptides, enzymes, and amines. One of the important components of bee venom is the phospholipase A2 enzyme, which is considered an important honey bee venom allergen and is also used to treat some diseases. This enzyme is found in other insects, arachnids, snakes, and mammalian cells, and its function is the hydrolysis of the second ester bond of glycerophospholipids and the release of fatty acids and lysophospholipids. Although transient transfection can produce recombinant proteins, stable cells are more suitable for high-scale production with economic efficiency.

The present study created a stable cell line to produce recombinant phospholipase A2 from honey bee (Apis mellifera) venom.

Plasmid cloning DNA vector containing phospholipase A2 gene was prepared by Macrogen Company. The recombinant plasmid was transferred to Chinese hamster ovary cells by heat shock method, and gene expression was carried out in a HamsF12 culture medium containing neomycin antibiotic. After increasing polyclonal strains containing plasmid, monoclonal clones were selected by limiting dilution. Then, monoclonal clones were propagated, the soup of the selected cells was collected and concentrated, and the protein expression was checked by sodium dodecyl-sulfate polyacrylamide gel electrophoresis test.

The results of electrophoresis, which was performed to confirm the expression of the phospholipase A2 gene in the cell soup, showed a band with a molecular weight of 20 kilodaltons, which confirms the creation of a stable cell line for the production of recombinant phospholipase A2 honey bee venom.

After the transient transfection of the plasmid containing this gene, several cells undergo recombination due to having repair mechanisms and putting the desired gene along with the antibiotic resistance gene in their genome. These cells can be selected and propagated by adding antibiotics to the culture medium.