Dissertation > Excellent graduate degree dissertation topics show

Cloning and Expression of the Heat Shock Protein 70 Gene from Mantichorula Semenowi

Author: TangZuo
Tutor: RenGuoDong;LiuFengSong
School: Hebei University
Course: Zoology
Keywords: Mantichorula semenowi hsp70 gene hsc70 gene β-actin semi-quantitative RT-PCR prokaryotic expression preparation polyclonal antisera Western Blot
CLC: Q51
Type: Master's thesis
Year: 2009
Downloads: 23
Quote: 0
Read: Download Dissertation


Mantichorula semenowi Reitter,1889 (Coleoptera:Tenebrionidae) is an endemic species distributed in the floating dune of Southeast Alxa, China and South Mongolia which adjoins China. The species evolved special physiological and ecological characteristics of adaptation under environmental stress. Its imago can survive on the surface of dune with extreme temperature as high as 70℃and use the microhabitat to produce offspring. The present results provide new insights into mechanisms used by M. semenowi to adapt to stressful environments.This thesis contains 3 parts.1.The cloning and expression analysis of Mshsp70 and Mshsc70 from M. semenowi. The cDNA was cloned using RT-PCR and RACE methods.2.The cloning and expression analysis ofβ-actin from M. semenowi. The cDNA was cloned using RT-PCR and RACE methods, and detected reliability ofβ-actin genes as internal control within insects after heat shock. To establish a stable expression in vivo geneticβ-actin steward for the internal control semi-quantitative RT-PCR method. By this method of heat shock the gene mRNA expression level of Mshsp70 and Mshsc70 relative quantitative were researched in M. semenowi.3.The Mshsp70 prokaryotic expression vector was constructed and in DE3 induction expression, to purify the protein making antibody in rabbit.The results are offered as follows:(1) The full-length cDNA of Mshsp70 is 2137 bp, containing a 3’-untraslate region of 168 bp, which encords a deduced 649 amino acid peptide with a predicted molecular mass of 71.34 KD, the result of sequence alignments analysis showed that this amino acids shares 80% identity with Harmonia axyridis and protein shares 85% identity with Tribolium castaneum.(2) The cDNA of Mshsc70 is 1908 bp, containing a 3’-untraslate region of 100 bp, which encords a deduced 601 amino acid peptide with a predicted molecular mass of 57.85 KD, the result of sequence alignments analysis showed that this nucleotide and protein shares 80% and 85% identity with Tribolium castaneum respectively.(3) The full-length cDNA ofβ-actin is 1372 bp, containing a 5’UTR of 66 bp and 3’UTR of 175 bp. The open reading frame ofβ-actin is 1131 bp, which encords a deduced 376 amino acid peptide. The result of sequence alignments analysis showed that this nucleotide share 96-99% identity with other animals. The sequence of theβ-actin was submitted to GenBank and assigned the accession number EU825991. Profiles ofβ-actin expression in different recovering time after heat shock were expressed similarly and not significantly different with that of the unstimulated control, suggesting an ubiguitous expression pattern can be used as internal control in gene mRNA expression level.(4) Semi-quantitative RT-PCR analysis showed that 1 h heat shock treatment at 42℃caused rapid increase of Mshsp70 mRNA, which reached maximum levels at the end of the heat shock treatment, and decreased gradually after being moved to room temperature. Expression of Mshsc70, however, was inhibited after heat-shock treatment:during recovering 2-4 h, the expression levels were only little times that of the control.(5) The Mshsp70 prokaryotic expression vector was constructed by prokaryotic vector pET-DsbA, and the recombined plasmid was transfected into DE3. After induction by cultivating 37℃, eventually concentration IPTG 1mM, the specific expression of the DsbA-fused protein was detected by SDS-PAGE. The result of SDS-PAGE showed that the molecular weight of the recombined protein is approximately 59.4 KD, which is consistent with its theory molecular weight. Using His affinity chromatography purified get fusion protein, to preparation HSP70 antibody, after western blot detected and expected the same size of specific bands.

Related Dissertations

  1. Expression of D-AtCGS in E. Coli and Preparation of Polyclonal Antibody Against D-AtCGS,Q943.2
  2. Cloning and Expression Analysis of GPx, GST and SAHH Genes in Chlamydomonas Sp. ICE-L from Antarctica,Q943.2
  3. cDNA Cloning, Expression of vp5 and vp7 Genes and Subcecullar Localization of VP5 and VP7 Proteins in Grass Carp Reovirus,S941.41
  4. Cloning, Expression of vp6 and ns38 Genes and Immunogenicity of VP6 and NS38 in Grass Carp Reovirus,S941.41
  5. Functional Analysis of Proteins Encoded by RNA2 of Wheat Yellow Mosaic Virus,S435.121
  6. Comparative Study on Reproductive Biological Characteristics of Helicoverpa Armigera and Helicoverpa Assulta (Lepidoptera:Nuctuidae),S433
  7. Cloning and Expression Analysis of Scavenger Receptor Class B Type Ⅰ and Antifreeze Proteinstype Ⅱ Genes in Lutjanus Sanguineus,S917.4
  8. Identification of B.melitensis 16M Protein Immunodominant Antigens,S852.61
  9. Isolation and Identification of Infectious Bronchitis Virus and Sequence Analysis of Its S1 Gene and N Gene,S852.65
  10. Isolationand Identification of Porcine Parvovirus and Parts of Its Biological Characteristics,S852.65
  11. Prokaryotic Expression and Purification of Human Beta-Defensin-9,Q78
  12. Molecular Cloning, Prokaryotic Expression and Characterization of a Novel Trypsin Inhibitor in the Frog, Lepidobatrachus Laevis,Q78
  13. Biochemical Characterization and Molecular Modification of a Novel Marine Mud-derived Pyrethroid Hydrolase,X172
  14. Comparative Study on the Host Choice Mechanism of Helicoverpa Armigera (Hübner) and H.assulta (Guenée),S435.622.3
  15. cDNA-cloning and Expression analysis of Phosphate Stress Induced Purple Acid Phosphatase Genes and transcription Factor Genes from Seeding Wheat,S512.1
  16. The Expression of Rabbit Bordetella Bronchiseptica PRN Protein and the Study on Its Immunoprotection,S855.12
  17. Cloning and Expression of Fimn Gene of Bordetella Bronchiseptica in Rabbitry and Establishment of Rapid Detection Method,S858.291
  18. Identification of Salt Tolerance and Function Analysis of NHX1 Gene in Glycine Max, Glycine Soja and Their Hybrid Seedlings,S565.1
  19. Cloning and Expression Analysis of Cold-Related Genes of Ammopiptanthus Nanus,S793.9
  20. Primary Study on CLC1 Gene Identification and Its Function in Glycine Max, Salt-born Glycine Soja and Their Hybrid,S565.1
  21. Cloning and Expression Analysis of NCCRP-1 AND IL-10 Genes in Grass Carp (Ctenopharyngodon Idellus),S917.4

CLC: > Biological Sciences > Biochemistry > Protein
© 2012 www.DissertationTopic.Net  Mobile