Background:
Tetanus is an infectious disease caused by clostridium tetani secreting tetanus toxin in anaerobic
environment. The fragment C of Tetanus toxin (TTc) has been widely studied as a candidate vaccine to replace the existing tetanus
toxoid vaccine.
Objective:
In this study, we established a simple method to purify recombinant protein TTc with ion-exchange chromatography from
Escherichia coli expression systems.
Methods:
The TTc gene sequence was cloned into pET26b (+) vector and transferred to E. coli BL21 (DE3) for expression. The
fermentation conditions (IPTG concentration, Induction temperature, Induction time) were optimized to obtain more soluble proteins.
The soluble proteins were purified by Anion exchange chromatography and Cation exchange chromatography. The sequence of
columns in the purification process was discussed. Finally, the stability of purified TTc protein were determined, the secondary
structure of the purified TTc protein was determined by circular dichroism. The molecular weight of the purified TTc protein was
determined by liquid chromatograph-mass spectrometer. Furthermore, we verified the immunogenicity of the purified protein in mice.
Results:
The purity of TTc improved from 34% to 88 % after the first anion exchange column, and the final yield of recombinant TTc
(purity > 95 %) can reach 84.79 % after the following cation exchange chromatography. The recombinant TTc had a molecular weight
of 51.737 KDa, was stable at 4 °C and weak alkaline environment, was a β-sheet secondary structure, and had strong immunogenicity.
Conclusion:
The purification method we developed might be an efficient method for the industrial production of tetanus recombinant
TTc vaccine.
AbstractTo study the extraction technology of polysaccharides (AAP) from Chinese herbal medicine formula and its mechanism of delaying aging. First, L9(3)4 orthogonal test was used to optimize the optimal enzyme-assisted extraction parameters of polysaccharides. And the anti-aging effects was evaluated by detecting mitochondrial function, protein, DNA, adhesion molecules and cell cycle in aging rats. The optimal extraction process parameters were the cellulase concentration of 1.5%, the pH at 5, the enzyme temperature at 50°C and the extraction time of 180 min. The anti-aging results showed that AAP can effectively increase the activities of malate dehydrogenase, succinate dehydrogenase and superoxide dismutase. It also can decrease the activity of monoamine oxidase and methane dicarboxylic aldehyde levels in the brain tissue. Meanwhile, the polysaccharides enhanced telomerase activity while reduced p16 protein expression of the brain mitochondria. In addition, the polysaccharides continued to improve heart damage and significantly lessen mitochondrial DNA concentrations. For a certain period of time, it also enhanced the activity of superoxide dismutase, reduced glutathione, glutathione peroxidase and decreased protein carbonyl and methane dicarboxylic aldehyde content of kidney in D-galactose-induced aging rats. Furthermore, the polysaccharides restored the number of cells in the peripheral blood lines and BMNC through inhibiting the drop of the number of red blood cells, white blood cells, platelets in the peripheral blood and bone marrow mononuclear cell of the aging rats. At the same time, AAP accelerated G1 phase cell to enter S phase in cell cycle in aging rats. Our research suggests that the polysaccharides may be a potential anti-aging agent and can be further developed as a functional food or new drug to delay aging or treat aging-related diseases.
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