Dissertation > Excellent graduate degree dissertation topics show

Expression of Human HSA-IFNα-2b Gene in Pichia Pastoris and the Analysis of Expression Stablity

Author: ZhangWuKui
Tutor: SongLiHua；FanQingLin
School: Anhui Medical University,
Course: Biochemistry and Molecular Biology
Keywords: electroporation rHSA-IFNα-2b Mutphenotype Wish-VSVsystem ELISA
CLC: Q789
Type: Master's thesis
Year: 2006
Downloads: 147
Quote: 0
Read: Download Dissertation

Abstract


Objective The competent Pichia pastoris was transformed by the recombinant plasmid pPIC9k-HSA-IFNα-2b which was cut with restriction endonuclease SalI through electroporation. Then the target protein was induced to express by methanol. We made the preliminarily study on the expression conditions and the genetic stability of the positive transformants. Methods The integrate gene pPIC9k-HSA-IFNα-2b was identified by digesting with restrictive endonuclease EcoRI/BamHI. Then the recombinant plasmid pPIC9k-HSA-IFNα-2b was digested with restrictive endonuclease SalI. The lined recombinant plasmid DNA was transformed into the competent Pichia Pastoris SMD1168 through electroporation, then the Pichia Pastoris integrants was analyzed with PCR method and screened for the Mut phenotype of positive transformants on MM and MD plate. The positive transformants were induced to express by methanol and the expressed supernatant was identified by Western-Blot. The expression level of positive transformants was identified by ELISA and the level of its G418 resistence was measured on YPD-G418 plate. The anti-virus activity of the expressed rhHSA-IFNα-2b was measuredby Wish -VSV system. We studied the effect of methanol concentration and OD600 value on the expression level of the positive transformants. Finally, we analyzed the genetic stability of the positive transformants. Results The lined recombinant plasmid DNA successfully transformed into the competent Pichia pastoris SMD1168 and integrated into SMD1168 chromosome through electroporation and the target protein was expressed in positive transformants and secreted into medium, which was induced by methanol. The expression level reaches as high as 300mg/L in medium through ELISA and the specific activity was

Related Dissertations

  1. Study on Bispecific Monoclonal Antibody to Pesticide Imidacloprid and Parathion-Methyl,S482.2
  2. Research of the Correlation About the Expression of Mfap4 in Liver Fibrosis Tissues and Concentrations of Mfap4 in Peripheral Blood Associated with Liver Pathological Grade,R575.2
  3. Electroporation Assisted Surface-enhanced Raman Spectroscopy for Living Cells,R318.51
  4. Establishment of ELISA for Detecting Cd-MT of Pteria Penguin and Preliminary Study of ELISA-kit,X835
  5. Establishment of Inderect ELISA for Detection of Antibodies Angainst STLV-1 and Preparation of Hybridoma Cell of Monoclonal Antibodies,R373
  6. Studies on Dynamic Regularity of Antibody Against G. Anatis and Development of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against G. Anatis Lps,S858.32
  7. Purification of Human Respiratory Syncytial Virus Fusion Protein with Immunomagnetic Microsphere Technology,R373
  8. Development of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against Lps Antigen of B. Melitensis,R392
  9. Construction and Application of the Integrated Gene Containing Multi-Mimotopes and VP2 of Infectious Bursal Disease Virus,S852.65
  10. The Expression of CPV-2 VP2 Gene in SF9 and Development of Indirect Elisa for Serum Antibodys,S852.65
  11. Improvement in Electroporation Transformation Efficiency of Gene for Bacillus Subtilis with Trehalose,Q78
  12. The Research on Enzyme-Linked Immunosorbent Assay for Pretilachlor and Acetochlor,S482.4
  13. Production of Monoclonal Antibodies Against Thiabendazole and Development of Heterologos Elisa,S482.2
  14. The Research on Enzyme-Linked Immunosorbent Assay for Imidaclothiz,S482.3
  15. Roles of MMP-7 and Lysozyme in the Pathogenesis of Ulcerative Colitis Induced by DSS in Balb/c Mice,S858.91
  16. Epidemiologic Studies on Duck Circovirus Infection in East China and Development of Monoclonal Antibody Against Duck Circovirus,S858.32
  17. Establishment of ELISA Based on Recombinant Protein NS1 of Swine Influenza Virus and Nuclear Localization of SIV NS and NP Proteins,S858.28
  18. The Establishment of the Diagnostic Method for Detecting Antibody Against Avian Leukosis Virus Subgroup J and Function Analysis of the Long Terminal Repeat,S858.31
  19. Development of Mcabs Against H9 AIV and Establishment of Antigen Capture Elisa for Detecting H9 Avian Influenza Virus,S855.3
  20. The Establishment and Application of cPL Double Anti-Body Sandwich Elisa Method for the Diagnosis of Canine Acute Pancreatisis,S858.292
  21. The Expression of Rabbit Bordetella Bronchiseptica PRN Protein and the Study on Its Immunoprotection,S855.12

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