Dissertation > Excellent graduate degree dissertation topics show

Fusion Expression and Penetrating Ability Study of Cell Penetrating Peptide TAT and Induced Pluripotent Stem Cell Related Transcriptional Factors

Author: WangJun
Tutor: JinJian
School: Jiangnan University
Course: Microbial and Biochemical Pharmacy
Keywords: TAT iPS inclusion body purification
CLC:
Type: Master's thesis
Year: 2013
Downloads: 42
Quote: 0
Read: Download Dissertation

Abstract


The generation of induced pluripotent stem cell (iPS) has become the main focus of stemcell research since somatic cells were successfully reprogrammed into stem like cells usingectopic expression of different reprogramming factors: Oct4, Sox2, Klf4, c-Myc. However, theinsertion of exogenous genes into genome increases the risk of tumorigenicity, which mayhave an effect on the clinical application of iPS technology. Therefore, protein-based methodprovides an alternative and ideal technology for generating therapeutical iPS. In this study, weexploited the cell penetrating ability of TAT to obstain TAT-Oct4, TAT-Sox2, TAT-Klf4,TAT-c-Myc and TAT-Nanog fusion proteins as a basis for generating safer iPS.TAT-Oct4, TAT-Sox2and TAT-Klf4target genes were amplified using PCR technology.TAT-c-Myc and TAT-Nanog were amplifed using overlapping PCR. All PCR products weredigested with Nco I and Xho I and then inserted into the pET28a expression vector. Therecombinant expression plasmids were verified by sequencing. Then the recombinant plasmidwas transformed into BL21(DE3) expression strain. The SDS-PAGE analysis showed thatrecombinant strains respectively habouring TAT-Oct4, TAT-Sox2, TAT-Klf4, TAT-c-Myc andTAT-Nanog were obstained.The target proteins TAT-Oct4, TAT-Sox2, TAT-Klf4, TAT-c-Myc and TAT-Nanog weremainly expressed with insoluble inclusion bodies in E.coli. And after inclusion bodieswashing and Ni sepharose purification, the purity of each target protein was more than90%.The addition of chemical additives such as arginine, glycerol and GSH/GSSG during ureagradient dialysis improved the average refolding yield of each protein which was8.8%,51.0%,47.9%,67.2%,8.5%respectively.Immunocytochemistry analysis showed TAT-mediated iPS related factors couldefficiently penetrate target cells. And the optimal concentration for penetrating cells was6μg/mL,8μg/mL,8μg/mL,8μg/mL and4μg/mL individually. In order to detect the relativequantity of each protein in cells, Flow cytometry analysis showed the fluorescence intensityvalue of each protein was37.5,44.5,38.7,44.7,64.7and the relative fluorescence intensityratio was16.6%,20.6%,17.3%,20.7%and33.2%.

Related Dissertations

  1. Study on Protective Effects of Apple Polyphenol on Immune System Injury Caused by γ-Ray,S661.1
  2. Study on Wastewater Purification Effect of the Free Surface Wetland with Different Substrates and Plant Combination,X703
  3. Penicillium Expansum TS414 Lipase in Pichia Pastori: Expression, Purification and Enantioselective Esterification of (R, S)-naproxen,Q814
  4. Studies on Fermentation Optimization, Purification and Enzyme Characteristics of Lipase from Aspergillus Oryzae FS-1,TQ925.6
  5. Studies on Isolation, Purification and Chemical Structure for Polysaccharides from Camellia Chrysantha(Hu) Tuyama,S567.19
  6. Research of Glycosaminoglycan from Perna Virids Effect on Blood Lipid,R285.5
  7. Purification of Endogenous Proteases from (Litopenaeus Vannamei) Shrimp Head and Their Enzymology Properties,S985.21
  8. Under microaerobic conditions closed calcium carbide furnace exhaust COS gas adsorption purification,X781
  9. Studies on Extraction and Analysis of Fructooligosaccharides in Smallanthus Sonchifolius,TS255.1
  10. Experiment Study of the Biogas Purification Process by Zeolite Filling Washing,TQ221.11
  11. Optimization of Fermentation Conditions, Purification, Cloning and Expression of a Cold-active Lipase from Pseudomonas Sp.RT-1,TQ925
  12. Extraction, Purification and Antifungal Activity of Anthocyanins in Drop Fruits of Chinese Baberry,TQ914.1
  13. Preliminary Research on Characterization of Thermophilic Protease Pasey and Helicase Htc16,Q814
  14. Study on the Separation and Purification Way of Arctiin from Fructus Arctii and the Effect of Arctiin on the Growth of Mice C2C12 Cells,S853.7
  15. Purification of Human Respiratory Syncytial Virus Fusion Protein with Immunomagnetic Microsphere Technology,R373
  16. Prokaryotic Expression and Purification of Human Beta-Defensin-9,Q78
  17. Screening of Bacterial Strains with High α-Galactosidase Activity and Its Gene Cloning, Expression, Purification and Characterization,TQ925
  18. Study on Extraction, Isolation and Antioxidant Activity of Lignans of Sesame Cake,TS229
  19. Study of Isolation and Identification on Antimicrobial Substances Produced by Lactobacillus Acidophilus NX2-6,TS201.3
  20. Study on Antioxidant Activities and Lipase-catalyzed Transesterification of Lard to Produce L-ascorbyl Fatty Acid Esters,TS202.3
  21. Isolation, Purification and Antioxidant Activity of Proteins from Chinese Yam,TS255.1

CLC: >
© 2012 www.DissertationTopic.Net  Mobile