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Genetic Cloning and Protein Purification of Mouse Methionine Sulfoxide Reductase A
China (IFMSA-China) - Wuhan University School of Medicine, Wuhan
Department of Biochemistry and Molecular biology
Type of Research Project
- Basic science
What is the background of the project?
Atherosclerosis is a common cardiovascular diseases, which is characterized by the accumulation of lipid on the vascular wall. In recent decades, it is regarded as a chronic inflammatory process related to multiple immune cells and oxidative stress. Therefore, new therapeutic drugs may be explored from the perspective of anti-oxidation and anti-inflammation. Methionine sulfoxide reductase areduces(MsrA), catalyzing the reduction of methionine-S-sulfoxide to methionine, plays a vital role in the maintenance of redox homeostasis in cells. However, the effects of MsrA on the metabolic diseases, such as atherosclerosis are still unknown now. Base on our previous study, we would like to produce purifiy MsrA protein by genetic cloning for in vivo study in mice subsequently.
What is the aim of the project?
To gain high-purified MsrA proteins for exploring its in vivo effects on atherosclerosis in mice laterly.
What techniques and methods are used?
1)Gaining the cDNA sequence of MsrA from mouse liver, and designing primers for generation of a recombinant pET28a-MsrA vector. 2) Affinity chromatography using the Ni-NTA beads to purify MsrA proteins 3) The purified protein was analyzed by SDS-PAGE and mass spectrometry.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
The student will participate in the whole project. Firstly, the student should download the MsrA sequence from pubmed or other database. Then, he/she need to generate a pET28a-MsrA vector and transfer the plasmid into E.coli. After that, MsrA proteins will be purified by Ni-NTA beads, owing to the high affinity of nickle ions and histidines. Finally, The purified protein was analyzed by SDS-PAGE and mass spectrometry.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
There will be several theoretical teaching provided for the student (preliminary readings, lectures, seminars), the content of teaching includes DNA cloning, affinity chromatography and protein electrophrosis, and lectures will be present by Professor and her postgraduates.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation - The student will prepare a scientific report - No specific outcome is expected
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Basic biochemistry knowledge and experiments skills Subjects passed: biochemistry Previous experience with: basic biochemistry experiments
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Students in biomedical fields
- Xu YY#; Du F#; Meng B; Xie GH; Cao J; Fan D; Yu H*. Hepatic overexpression of methionine sulfoxide reductase A reduces atherosclerosis in apolipoprotein E-deficient mice. J Lipid Res 2015;56(10):1891-1900.
- Wu Y; Xie G; Xu Y; Ma L; Tong C; Fan D; Du F#; Yu H#. PEP-1-MsrA ameliorates inflammation and reduces atherosclerosis in apolipoprotein E deficient mice. J Transl Med 2015;13:316.
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