Dissertation > Excellent graduate degree dissertation topics show

Fusion Expression of Six Temporin-1CEa Genes from the Skin of Chinese Brown Frogs, Rana Chensinensis Repeat in Tandem

Author: ZhangLiFang
Tutor: ShangDeJing
School: Liaoning Normal University
Course: Genetics
Keywords: Antimicrobial peptide Temporin-1CEa Gene recombination Fusion expression
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 90
Quote: 1
Read: Download Dissertation

Abstract


Antibacterial peptides (ABPs),small molecule peptides encoded by the specific gene, have broad-spectrum antimicrobial activity. Amphibian skin peptides are a kind of ABPs considered to be a main element of innate immunity, which are also the weapon for fighting back the pathogenic bacteria. For the emergence of multiple-antibiotic resistant strains, ABPs from amphibian skin play an important role in the development of novel antibiotics because their broad-spectrum antimicrobial activities and specific antimicrobial mechanism. However, the isolation and purification of natural bioactive peptides from amphibian skin is too difficult to meet the needs of large-scale production. So, it is an inevitable choice to produce bioactive peptides by the gene expression in prokaryotes or eukaryotes. Amphibian skin recombination peptides can not be expressed directly in prokaryotic expression system because the high antimicrobial activity. Therefore, the fusion expression system, or expression in eukaryotic cells for recombination anti-bacterial peptides expression often is used.In this reseach ,we use recombinant technology and the fusion expression system to express six temporin-1CEa genes from the skin of Chinese brown frogs. Based on the amino acid sequence of temporin-1CEa,We synthesize temporin-1CEa gene sequence .We use PCR method to connect the six of temporin-1CEa genes, of which each gene is preceded by ATG codon. Lastly, at the 5 ’and 3’ end, two restriction sites of Sma I / Hind III were added, respectively. The 339bp fragment was constructed into the expressive vector pET-42a(+) for the expression of GST-temporin-1CEa fusion protein after six of temporin-1CE gene were synthesized in tandem.The sequencing analysis confirmed that the six of temporin-1CEa genes had been correctly inserted into vector pET-42a(+). The recombinant vectors were transformed into E.coIi BL21.The GST-temporin-1CEa fusion proteins expressed in the host bacteria under the IPTG induction,,and analyzed with SDS-PAGE. Different induced temperature, pH, IPTG concentrations and the induction time were screened to determine the optimum inducing conditions. At last, we choose the condition as following: after bacteria growing in LB medium of pH7.2 to saturation, induced recombinant protein for 4h at 30℃with 0.8mM IPTG. The experimental results showed that the recombinant protein molecular weight is 43KD, which occupy 10.8% content of total bacterial protein and present in the form of soluble protein in bacterial lysates. Fusion protein was purified with GSTrap FF affinity chromatography and HisTrap HP affinity chromatography. The purity of fusion protein is about 60% after purifying. After the fusion tag was cleaved by factor Xa, the antibacterial activity was tested. The construction of prokaryotic expressive plasmid of temporin-1CEa gene establishes a solid basis for further studying the amphibian skin antibacterial.

Related Dissertations

  1. Development of Inactivated Vaccine Against Streptococcus Equi Zooepidimicus in Whole Cell Binding M-Like Protein Subunit Vaccines,S858.28
  2. Effects of Lactobacilli on the Gene Expression of Antimicrobial Peptide AvBD9 in Chicken Intestinal Epithelial Cells,S831
  3. The Purification and Isolation of Antimicrobial Peptides from the Grass Carp Intestine and the Research on Physicochemical Properties,S917.4
  4. Separation, Purification and GST Fusion Expression in Escherichia Coli of Antimicrobial Peptides from Pseudosciaena Crocea,S917.4
  5. Expression and Activity Assay of Amylomaltase in Pichia Pastoris,Q78
  6. Studies on the Molecular Biological Characteristics and Phylogenetics of H6N1 Subtype AIV Strains Isolated from Mallard,S852.65
  7. Construction and Identification of Recombinant Adenovirus Vector Containing rhGM-CSF Gene,R346
  8. Study on the Expression and Bioactivities of Porcine Interleukin-2/6 Fusion Protein in Vivo and in Vitro,S858.28
  9. E. Coli Fusion Expression of β-mannanase and Characterizing,Q78
  10. Study on the Construction, Expression and Bioactivity of Chicken Interferon Alpha/Chicken Interleukin-2 and ChIFN-α-Linker-ChIL-2 Fusion Protein,S852.5
  11. Preparation of Recombinant Antimicrobial Peptide Fowlicidin-1 and Its Antimicrobial Activity Analysis,Q78
  12. Cloning and Expression of Rat Gene DJ-1,Q78
  13. Expression of Bombyx Mori ABP Fusion Protein Indusing by Lactose,Q786
  14. Study on Induced Immune Response to Microbial Infection of Chrysomya Megacephala Lavrae,S476
  15. Expression of Antimicrobial Peptide from Rana Catesbiana in Pichia Yeast and Its Biological Activity,Q78
  16. Molecular Cloning and Expression Vector Construction of the mgrA Gene and Expression and Purification of the Fusion Protein in E.coli,Q78
  17. Isolation and Purification of Antibacterial Polypeptide from Cow Uterine Endometrium,S823
  18. Screen and Study of Novel Antimicrobial Peptide Genes from Penzer Crucian,Q78
  19. Construction of Prokaryotic Expression Plasmid with Two Subgroups of Human ndrg2 Gene and Expression and Purification and Anti-tumor,R730.5
  20. Expression in Pichia Pastoris of Antimicrobial Peptides from the Skin of Rana Dybowskii and Its Antimicrobial Activity,Q78

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