Dissertation > Excellent graduate degree dissertation topics show

Human Granulocyte-Macrophage Colony Stimulating Factor (hGM-CSF) Gene Expression in Anabaena sp.PCC7120 and the Reconstruction of Its Expression System

Author: JinRui
Tutor: WangQuanXi；ShiDingJi
School: Shanghai Normal University
Course: Hydrobiology
Keywords: Human Granulocyte-Macrophage Colony Stimulating Factor Anabeaba sp. PCC 7120 triparetal conjugative transfer transgenic cyanobacteria Shine-Dalgarno(SD) sequence homology recombination method
CLC: Q943.2
Type: Master's thesis
Year: 2006
Downloads: 105
Quote: 0
Read: Download Dissertation

Abstract


Human Granulocyte-Macrophage Colony Stimulating Factor (hGM-CSF) is the first haematopoietic cytokines that has been cloned and been utilized by recombinant DNA technology. It has potent effects in stimulating proliferation, maturation and function of haematopoietic cell. It occupied large percent of recombinantion medicine market. In the meantime, commercial produced hGM-CSF usually use E. coli to be the host. For its disadvantages of overexpression and endotoxin, people try to express hGM-CSF in many other hosts just as yeast, mammal, hexapod, etc. But the results have not been optimized. Cyanobactreria as the potential candidate host to produce genetic engineering medicine have simple structure and makes them be a potentially powerful model system of modern biotechnology, and they provide advantageous hosts to produce organic substances. Based on these advantages, we attempt to express hGM-CSF gene in cyanobacterial cell.In this article, base on successfully expressed hGM-CSF in Anabaena sp. PCC 7120, we modified hGM-CSF gene by add shine-dalgarno(SD) sequence in N-terminal and adjusted the distance between it and ATG which affects the efficiency of translation. Then inserted it into downstream of the promoter PpsbA, ligated it with pDC-08 to contruct the shuttle expression vector, pDC-GMs. Then transformed it into Anabaena sp. PCC 7120 by the triparental conjugation transfer method. Gained transgenic sp. PCC 7120 harboring pDC-GMs by screening with neomycine. Then optimized the culture conditions to found the restrict curver factors. And for the reason of the prophase shuttle vectors which we constructed have the unstable disadvantage, we cloned glnA gene which encoding glutamine synthetase by TD PCR in Anabeaba sp. PCC 7120 as a integrative platform. Then, an integrative expression vector pRG-GM which contained hGM-CSF gene and endogenous promoter PpsbA was constructed. It provides reference to improve the expression level and the stability of foreign hGM-CSF gene in Anabeaba sp. PCC 7120.The contents and results of this experiment were as follows:1. We add -GGAGAG- SD sequence in hgm-CSF N-terminal and adjusted the sequence between SD and ATG into 6bp by PCR. Then inserted it into downstream of the strong promoter, PpsbA of shuttle vector pRL-439, then ligated it with pDC-08 to contruct the shuttle expression vector, pDC-GMs. Then transformed it into Anabaena sp. PCC 7120, by the triparental conjugation transfer method. Screening of positive clones were performed on BG-11(-N) agar plates supplemented with neomycine. PCR amplification of

Related Dissertations

  1. Construction of Recombinant Adenovirus Expression Vector Carrying GM-CSF and BZLF1 Gene,R373
  2. Construction and Expression of DNA Vaccine Vector pVAX1-CAIX-GM of Renal Cell Carcinoma,R737.11
  3. The Protective Effects of Recombinant hGM-CSF in Lactococcus Lactis on Bowel Injury of Rats Induced by TNBS,R574
  4. Expression, Purification and Characterization of hGM-CSF Using B. Mori Pupae as a Bioreactor,Q786
  5. Strategies to Improve the Expression Level of Human Granulocyte-Macrophage Colony Stimulating Factor in Plants,Q943.2
  6. Cultivation and Photosynthesis of Transgenic Synechococcus sp. PCC7002 with hTNF-α Gene,Q945
  7. The Investigation on Cloning and Expression Regulation of egfp Gene in Synechocystis sp. Strain PCC 6803,Q943.2
  8. Expression of D-AtCGS in E. Coli and Preparation of Polyclonal Antibody Against D-AtCGS,Q943.2
  9. Cloning and Expression Analysis of GPx, GST and SAHH Genes in Chlamydomonas Sp. ICE-L from Antarctica,Q943.2
  10. An Approach for Identifying a Plant Resistance Gene Based on the Random Forest,Q943
  11. Cloning Andanalysis of NAC Transcription Factor Gene in Tobacco(Nicotiana Tabacum L.),Q943.2
  12. Bioinformatics Study of Motifs Within 3’UTR Region of Stress Response Genes and Functional Related Mirnas in Rice,Q943.2
  13. Molecular Clonging and Functional Analysis of Heat Shock Transcriptional Factor CarHSFB2 in Chickpea (Cicer Arietinum L.),Q943.2
  14. Expressive Characterization of Transcription Factors in Cotton Fibre Initiation and Cloning and Characterization of Three Transcription Factor Genes in Gossypium Hirsutum L.,Q943
  15. Cloning and Functional Analysis of DREB-Like Gene from Suaeda Salsa,Q943.2
  16. Transformation of Tobacco with Arabidopsis Thaliana MAP65-6 and TUA-6,Q943.2
  17. Effect of SO2 Exposure on DNA Methylation Polymorphism in Arabiadopsis Thaliana,Q943
  18. Introduction of Transgenic Plants Via Non-Tissue-Culture Methods,Q943.2
  19. Molecular Cloning and Prokaryotic Expression of Chalcone Synthase Gene in Fressia Hybrida,Q943.2
  20. The Arabidopsis STAR2 gene function analysis,Q943
  21. The Function Analysis of Ceramidase (CER) from Arabidopsis Thaliana,Q943
  22. Cloning and Sequence Analyses of CONSTAN (CO) and LEAFY (LFY) Homologous Gene of Dendrocalamus Xishuangbannaensis,Q943.2

CLC: > Biological Sciences > Botany > Plant Cell Genetics > Plant Genetic Engineering
© 2012 www.DissertationTopic.Net  Mobile