Mohsen Mehrvarz; Mohsen Fathi Najafi; Taghi Zahraei Salehi; Behjat Majidi
Abstract
Clostridium perfringens and novyi species are two important toxin-producing pathogens which pose a risk to the livestock health. Epsilon and alpha toxins are major toxins of these two pathogens, respectively. Advances in current vaccine industrialization lead to the utilization ...
Read More
Clostridium perfringens and novyi species are two important toxin-producing pathogens which pose a risk to the livestock health. Epsilon and alpha toxins are major toxins of these two pathogens, respectively. Advances in current vaccine industrialization lead to the utilization of toxin epitopes instead of the whole pathogen/toxoids to produce novel vaccines. In the present study, bioinformatics approaches were applied to design a fused protein containing both toxin fragments of interest with the highest antigenicity score for B-cells. To do so different specialized algorithms including I-TASSER, IEDB, ElliPro, PyDock and CLC Main Workbench were applied. The chimeric protein was successfully cloned, expressed, and purified using an immobilized-metal affinity chromatography for His-tagged proteins. During in vivo experiments on rabbits, the levels of immunization provided by the recombinant protein or native alpha and epsilon toxins were compared based on serological studies. Results indicated that the designed protein was able to stimulate effective immune responses against both alpha and epsilon toxins. This can be used as a proper strategy to design novel peptide-based subunit vaccines. Clostridium perfringens and novyi species are two important toxin-producing pathogens which pose a risk to the livestock health. Epsilon and alpha toxins are major toxins of these two pathogens, respectively. Advances in current vaccine industrialization lead to the utilization of toxin epitopes instead of the whole pathogen/toxoids to produce novel vaccines. In the present study, bioinformatics approaches were applied to design a fused protein containing both toxin fragments of interest with the highest antigenicity score for B-cells. To do so different specialized algorithms including I-TASSER, IEDB, ElliPro, PyDock and CLC Main Workbench were applied. The chimeric protein was successfully cloned, expressed, and purified using an immobilized-metal affinity chromatography for His-tagged proteins. During in vivo experiments on rabbits, the levels of immunization provided by the recombinant protein or native alpha and epsilon toxins were compared based on serological studies. Results indicated that the designed protein was able to stimulate effective immune responses against both alpha and epsilon toxins. This can be used as a proper strategy to design novel peptide-based subunit vaccines.
Jafar Vatandoost; Shadi Tirdad
Abstract
In the production of recombinant proteins, the selection of an expression system is very important. Although CHO cells are used as mammalian expression system, the ability of insect Schneider line 2 (S2) cells as a new expression system for the high production of many human proteins were confirmed. It ...
Read More
In the production of recombinant proteins, the selection of an expression system is very important. Although CHO cells are used as mammalian expression system, the ability of insect Schneider line 2 (S2) cells as a new expression system for the high production of many human proteins were confirmed. It is suggested that high copy number of an introduced gene and so high transcription in these cells can be regarded as one of the reasons for high expression of recombinant proteins. Therefore, the present study aimed to evaluate the correlation of recombinant human coagulation factor IX (hFIX) abundance and mRNA expression. The amount of hFIX mRNA by quantitative real-time PCR as well as hFIX protein in cell lysate and cultured media by Elisa was analyzed. The results of data analysis indicated 6 fold increases in mRNA level in S2 cells in comparison to CHO cells. Furthermore, S2 cell line indicated 5.5 and 7-fold increase in total and secreted protein level, respectively, compared to CHO cell line. The data demonstrated the correlation of mRNA and protein abundance and indicate that S2 cell lines are superior in producing the recombinant proteins.
