Dissertation > Excellent graduate degree dissertation topics show

Expression of Conserved Dopaminergic Neurotrophic Factor in sf9 Cells by Using Bac-to-Bac Expression System

Author: ZhangJun
Tutor: NiuChaoShi;FuXianMing
School: Anhui Medical University,
Course: Surgery
Keywords: bac-to-bac CDNF baculovirus transfer vector bacmid Parkin’s disease
CLC: R346
Type: Master's thesis
Year: 2011
Downloads: 6
Quote: 0
Read: Download Dissertation

Abstract


Objective: Use the Bac-to-Bac expression system to express recombinant protein CDNF in insect cells.Methods: Mouse total RNA was isolated using Trizol reagent then first-strand cDNAs were synthesized by reverse transcriptase.Full length (564bp) mouse CDNF was amplified by PCR. The PCR product was cloned into pFastBacHTb vector to create pFastBacHTb-CDNF vector which transformed into E.coli DH5a competent cells. Recombinant plasmid was selected by LB agar plates containing 100ug/ml ampicillin, and was further verified by PCR amplification, restriction analysis and sequencing. The constructed baculovirus transfer vector pFasBacHTb-CDNF was transformed into E.coli DH10 Bac competent cells. Blue/white selection was used to identify colnies containing the recombinant bacmid. Successful transposition to the bacmid will be verifid by PCR analysis using pUC/M13 forward and reverse primer. The recombinant Bacmid-CDNF DNA were transfected into Sf9 insect cells using CellfectinⅡreagent according to the manufacturer’s instructions. Infected and uninfected sf9 cells can be distinguished by morphology. Recombinant CDNF protein was prduced in sf9 insect cells after infection with recombinant baculovirus, Expression of recombinant protein was identified by western bolt.Results: Agarose gel electrophoresis after RT-PCR showed a 564bp band which consistent with the anticipation size. Recombinant plsmid pFastBacHTb-CDNF was constructed and successfully identified by double digestion of Xho I and BamHI restriction enzyme or single digestion of BamH I, PCR amplification and sequence. After the pFastbacHTb-CDNF transfer vector was transformed into DH10Bac E.coli, recombinant bacmid-CDNF was successfully verified by PCR amplification using pUC/M13 amplification primers and CDNF primer. Recombinant Bacmid-CDNF was isolated from over night cultures and transfected into insect cells sf9 with CELLFECTIN reagents. Uninfected cells continued to divide and form a confluent monolayer while infected cells stopped dividing and enlarged after 48-72 hours. Viral stock was isolated and storaged. Recombinant CDNF protein was induced to expression in sf9 cells. The molecular weight of the recombinant protein CDNF was approximately 21kD(plus with 3kD 6-His tag).Conclusion: The recombinant protein mCDNF was successfully expressed and purified using Bac-to-Bac expression system.

Related Dissertations

  1. Preliminary Study of Phosphorylation Effect on UGT1A3 Metabolism Activity Towards Drugs,R346
  2. Study on Genes Expression and Function of UDP-glucosyltransferases in the Silkworm, Bombyx Mori,S881.2
  3. Intracellular Trafficking and Secretion of Cerebral Dopamine Neurotrophic Factor in Neurosecretory Cells,Q42
  4. Detection of Infetious Flacherie Virus(Chinese Isolate) by Nested RT-PCR and Expression of the Viral RNA Polymerase,S884
  5. Expression of Pig Interferon a and Construction of Recombinant Bacmid,S852.5
  6. Construction of Baculovirus Transfer Vector Containing E2 Gene of CSFV and Researches about Apoptosis of Porcine Kidney Cells Induced by CSFV,S852.65
  7. Restructuring inorganic binding peptide binding metal oxide nanoparticles applied basic research,Q78
  8. UGT1A3 and UGT1A9: Heterogenous Expression, Flavonoids Glucuronidation and Functional Characterization of UGT1A3 Variants,R96
  9. Cloning, Expression and Functional Analysis of Olfactory Related Genes of the Rice Borer,S435.112
  10. Design and Expression of Hybrid Gene Encoding Thermostable Endoglucanase with High Activity and Property of Hybrid Enzyme,Q78
  11. Exogenous gene expression based on the baculovirus expression system,Q78
  12. Cocoonase silkworm gene cloning , expression and promoter activity analysis,Q78
  13. Expression Human Cytochrome P450 2C9 in Baculovirus-Insect Cell Expression System,Q78
  14. The Expression and Function Identification of Human Neuritin in Baclovirus Expression System,Q78
  15. Expression and Characterization of Human Recombinant CYP1A2 and CYP3A4,R96
  16. Study on Expression of the S Gene of Transmissible Gastroenteritis Virus and Biological Activation Analysis of the Expressed Proteins in E.coli,S852.65
  17. Sequence Analysis of Porcine Parvovirus SD-68 Strain and Its VP2 Gene Co-expression in Insect Cells with ORF2 Gene of Porcine Circovirus,S852.65
  18. Using real-time tracking technology Fluorescence Materials Visualization virus and cell interactions,Q939.4
  19. Tribolium iap gene cloning , expression patterns and molecular phylogenetic analysis,S379.5
  20. Expression and Bioactivity Evaluation of a Novel Aquaporin Gene from Photobacterium Profundum SS9,Q786

CLC: > Medicine, health > Basic Medical > Human biochemistry, molecular biology
© 2012 www.DissertationTopic.Net  Mobile