For Western blotting, after gel electrophoresis and after separation of proteins by SDS-PAGE, the proteins were transferred to a nitrocellulose membrane. After this, the gel was stained with coomassie blue R-250 and then destained. The proteins were mixed with 2X SDS-loading buffer (100 mMTris-HCl pH6.8, 20% glycerol, 4% SDS, 0.005% bromophenol blue and 200mM DTT) and heated at 85☌ for 5 min and applied for gel electrophoresis ( 9). SDS-PAGE was performed using a 12% polyacrylamide gel. SDS-PAGE and Western blotting were used to detect the induced polytopic-His6 protein. The induced cells were lysed in lysis buffer using sonication. The cells were harvested by centrifugation at 8000rpm for 5 min. The transformed cells were induced when OD600 of growing cells reached 0.6–0.8, by the addition of isopropyl-β-D-1-thiogalactopyranoside (IPTG) in a final concentration of 1 mM for 2–5 hours. The cultured cells were transformed with ligated pET28a vector. coli were cultured in Luria-Bertani (LB) broth medium, supplemented with kanamycin, at 37☌ with a shaking speed of 200 rpm. This protein was then expressed and purified, in order to be used for different medical applications.Įxpression of recombinant polytopic His6. gondii, in view of better stimulation of human immune system by in silico methods and constructed a DNA sequence, based on the indicated epitopes for the production of a poly-epitope protein. Herein, we selected the strongest epitopes of three surface antigens of T. The surface antigens are anchored to the plasma membrane and it appears that have significant exposed epitopes for immune systems, playing an important role in stimulation of host immune response and thereby can be used for applications of diagnosis and vaccine development ( 1, 7).
In the course of the acute disease, some of the SAG family members are clearly immunodominant and induce a strong immune response against the parasite, during early stages of infection. Heparin sulfate proteoglycan mediate attachment of the parasite to the host cell surface ( 6). SAG3, a surface protein similar to SAG1, in structure and associated with binding to the host cells. It has been shown that it is relevant in the host-parasite interface and interacts with both distinct innate and adaptive immune mechanisms ( 5). SAG2 is another attachment factor on the surface of the parasite, which is a serologically immunodominant protein. SAG1 has been demonstrated to be an important protein, in developing subunit vaccines and rapid diagnostic tests ( 4). SAG1 is one of the crucial immunodominant ligands in the invasion of the parasite into host cells. The surface of the parasite is covered with a type of glycosylphosphatidyl inositol (GPI)-anchored antigens, belonging to the surface antigens (SAGs) family ( 3). gondii possesses several antigens, which have been much used for designing a vaccine and prompt diagnosis ( 1, 2). Therefore, controlling approaches can minimize the parasite’s harm to people. This infection is costly to take care of and causing distress and significant problems for livestock husbandry. This obligate parasite causes death in immunocompromised and immusuppressed individuals, as well as in utero. It contains asexual phase in all hosts, including a sexual phase in the gut of the cat ( 1). Its complex life cycle involves definitive and intermediate hosts. gondii is a ubiquitous intracellular protozoan parasite that infects a wide range of warm-blood animals, including humans.
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