Dissertation > Excellent graduate degree dissertation topics show

Heterologous Expression of huGALNT3 and Prepartation of Anti-huGALNT3 Antibody

Author: KongYun
Tutor: WangPeng
School: Shandong University
Course: Microbiology
Keywords: N-acetyl- galactosamine COMT E. coli expression system Pichia pastoris expression system for antibodies
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 43
Quote: 0
Read: Download Dissertation

Abstract


Sugar chains and their conjugates have an important role in physiological and pathological significance. The sugar chains in the tumor cells compared with normal cells have changed significantly. No template, the synthesis of a sugar chain, but rather through a series of positioning orderly glycosyltransferase glycosyltransferase expression differences will lead directly to the changes in the structure of the sugar chain. Therefore, from the glycosyltransferase start essentially to clarify the role of the sugar chain in tumorigenesis and development process variation and regulation. N-acetyl-galactosamine-transferase 3 (UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 3, ppGalNAc-T3, GALNT3) is one of the members of the GalNAc transferase family. The family of catalytic O-sugar chain formed during the first reaction step, the upcoming GalNAc transfer to the polypeptide chain of the Ser / Thr residues. Differ between the family members in the active level and on the substrate selectivity. Wherein the activity of GALNT3 formation of O-sugar chain-rich mucin closely exception of the latter is a common phenomenon occurs in a variety of tumors of epithelial origin. This thesis is amplified from a human gastric cancer cell MKN45 GALNT3 the cDNA, the E. coli expression vector was constructed and expressed. Soluble and inclusion bodies in E. coli using two forms, successful implementation of efficient expression of the GALNT3 soluble region (GALNT3-sol), and purified. Found that after the activity was measured, and the expression of two forms the obtained GALNT3 no activity, the reason may be E. coli lack the mechanism of post-translational modifications in eukaryotic protein synthesis and GALNT3 is a complex glycoprotein of human-derived, the heterologous expression product is not to form active native conformation. Further, the present experiment also used GALNT3-sol prepared anti-GALNT3 antibody as an immunogen expressed in E. coli. The inclusion body degeneration refolded and purified after SDS-PAGE separation the plastic cutting immune BALB / C mice, respectively, to take the second and fourth immune serum by enzyme-linked immunosorbent assay (ELISA) to detect antibody price, free of anti-serum endpoint titers of 1:3200, and four free anti-serum endpoint titer of 1:25600. Then use the Western Blot method identified received antibody the positive antiserum capable of binding GALNT3-sol, to the color, negative control can not be combined with color. The above results show that this experiment made anti GALNT3-sol antibody with high titer and can be used for Western Blot analysis, and laid the foundation for the further development the anti GALNT3 monoclonal antibody and clinical materials. In order to obtain the active GALNT3 protein, the paper tried to expression in Pichia yeast (Pichia pastoris) GALNT3. Part of GALNT3 soluble protein gene (GALNT3-sol) was cloned on to pPIC9k plasmid was transformed into Pichia pastoris GS115 strain. PCR and gradient antibiotics (G418) after defects medium, yeast cells were screened for high-copy recombinant strains. Western Blot results prove successfully expressed GALNT3 in Pichia pastoris, the best expression of the conditions of 20 ° C, induced 72 hours after the methanol induced GALNT3 expression. The activity detection obtained GALNT3-sol, concluded that the the Pichia expression GALNT3-sol turn glycosylation activity, this sugar nucleotide selectivity or receptor polypeptide laid the foundation for further research GALNT3. In summary, the present paper in both microbial systems successfully expressed huGALNT3-sol, and the first use of the E. coli expression GALNT3-sol prepared efficient antibody, while the first time in microorganisms express active GALNT3 as production in the the yeast eukaryotic expression system for other human N-acetyl-galactosamine transfer enzymes, even other types of glycosyltransferases made possible. Expression of GALNTs active protein for in vitro starting O-glycosylation provide a new means to provide a framework to add a special biological function or activity of the compound on the drug-protein.

Related Dissertations

  1. Prokaryotic Expression and Renaturation of Streptavidin,Q78
  2. The Preparation and Chacterization of Arctium Lappa L. Oligosaccharide Liposome and Preliminary Study of Human α-1, 6 Fucosyltransferease,Q946
  3. Autographa Nuclear Polyhedrosis Virus (AcMNPV) pk-1 Gene Structure and Its Kinase Activity Analysis,Q78
  4. Clone and Expression of Bovine Recombinant Enterokinase Catalytic Subunit in Escherichia Coli,Q78
  5. Prokaryotic Expression of Major Epitope Domain of Porcine Parvovirus Structural Protein VP2 and Nonstructural Protein NS1 and the Establishment of Indirect VP2-ELISA,S852.65
  6. Throwing Alternaria yeast keto reductase (KR) expressed in Pichia pastoris and E.coli,Q786
  7. Expression of the Retinol-binding Protein and Interaction with Other Proteins,Q51
  8. Expression of SARS Coronavirus Spike Protein in E.coli and Bombyx Mori,R346
  9. Label genetically engineered protein cutting with enterokinase light chain gene cloning and expression,Q78
  10. Cloning and Expression of Genes Encoding Phospholipase A2 and Hyaluronidase of Honeybee bee Venom from Apls Cerana Cerana and A.Mellifera,Q785
  11. Cloning and Expression of the Venom Genes Encoding Secapin, Apamin, MCDP and Ag5 from Honeybee and Vespoidea Species,S891
  12. Expression, Purification and Function of Osteoprotrgerin,R341
  13. Study on Expression of HCV Structural Proteins and the Forming of HCV-like Particles in Insect Cells,R392
  14. Research on Combinatorial Regulation of Multiple Transcription Factors,Q78
  15. The Expression of Let-7 microRNA during the Process of the Mouse Embryonic Lung Development and the Construction of the Adenovirus Shuttle Plasmid,Q78
  16. Directed Evolution of Lipase from Penicillium Expansum FS1884 by Error Prone PCR,Q78
  17. Gene Prediction and Sequence Analysis of Insect OBP CSP and sid-1,Q78
  18. Expression of Glucose Oxidase Gene from Aspergillus Niger Z-25 in Pichia Pastoris,Q78
  19. Construction and Application of the Transgenic Yeast Strain Expressing Metallothionein,Q78
  20. Loach Dmrt1 gene cloning , expression and alternative splicing analysis,Q78
  21. Based on green fluorescent gene targeting studies of transgenic silkworm silk,Q78

CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
© 2012 www.DissertationTopic.Net  Mobile