RESUMO
Background: Since the mass production of recombinant proteins requires the development of stable cell lines which is a time-consuming complex process, the use of transient expression on a large scale can be a comparatively useful alternative. Although various cell lines have been used for the expression of recombinant proteins, only a limited number of cells enjoy a high transfection characteristic and the ability to adapt to serum-free suspension culture easily. In the present study, the S2 cells from Drosophila insect with the ability to grow in suspension and serum-free cultures were used for the expression of factor IX [FIX] using Transient Gene Expression [TGE] technique
Methods: Drosophila Schneider [S2] cells were seeded in special roller bottles, and then, the cells were transfected with pMT-hFIX plasmid employing the calcium phosphate co-precipitation method. The stable S2-hFIX cells were also seeded in special roller bottles, separately. After the induction, recombinant FIX was quantified in conditioned media employing an ELISA. Moreover, its functional activity was examined using an aPTT assay
Results: The results showed that the expression of FIX through TGE technology was 1.6 times as high as that obtained through S2-FIX stable cells. Furthermore, the comparison of the FIX expression in S2 cells through TGE techniques with that obtained in previous studies in HEK cells or CHO cells revealed that S2 cells were more efficient in terms of FIX expression
Conclusion: The S2 cells with the capability to grow in suspension and serum-free cultures are a suitable alternative for transient expression for the large scale production of protein
RESUMO
Background: Probiotics are beneficial and non-pathogenic microorganisms. Isolation of probiotic bacteria from traditional dairy products can not only lead to the isolation of probiotic bacteria with special characteristics, but it can offer a good approach for the mass production of traditional dairy products containing natural probiotic bacteria.
Materials and Methods: After collection of dairy products samples from different regions of Sabzevar, they were continuously cultured on the specific media of MRS and MRS Broth. Initial identification of isolates was performed by gram stain, motility test, nitrate reduction test, growth at 15 and 45 °C, growth at pH: 9/6 and fermentation capability of 11 different sugars. To identify desired strains more precisely, the 16S rDNA gene was amplified by PCR with specific primers and then sequenced and BLASTed. Acidic and bile salts conditions tolerance tests were performed for the final confirmation of desired strains.
Results: After continuous culture on specific agar media, 16 strains for further analysis were selected. In early identification of isolates by phenotypic methods, 14 strains were positive. To identify these strains more precisely, the 16S rDNA gene was amplified. Following molecular identification and sequencing of 16S rDNA gene, the BLAST sequence similarities were found with Lactobacillus planetarium. In addition, acidic and bile salts conditions tolerance tests showed that these bacteria had the best growth pattern at PH: 4 and they were able to grow in the presence of bile salts.
Conclusion: Biochemical results showed that the most common strains in the tested dairy products are Lactobacillus. These results also confirmed by the molecular tests. Acidic and bile salts conditions tolerance test, as a main characteristic of probiotic bacteria, showed that the strain was able to withstand these conditions.