Dissertation > Excellent graduate degree dissertation topics show

Construction of eukaryotic expression vector of human lactoferrin breast pBC1-hLF

Author: YuXiaoMing
Tutor: FanFeiYue；AnZuo
School: Peking Union Medical College , China
Course: Radiation Medicine
Keywords: human lactoferrin (hLF) mammary gland expression vector vector construction
CLC: R346
Type: Master's thesis
Year: 2006
Downloads: 75
Quote: 0
Read: Download Dissertation

Abstract


We constructed the middle vector pGEMEX-I-hLF to add the Sal I sites on the both terminals of the hLF cDNA, then digest the pGEMEX-I-hLF with restriction endonuclease Sal I to obtain the hLF cDNA and digest the vector pBCl with restriction endonuclease Xho I to obtain the liner vector. The expression vector could be constructed by ligation. Then it is used in the following experiments.The middle vector is easer to construct because of its size and directional cloning. But there are some problems in constructing the expression vector: (1) The transfection effect would be lower because the expression vector pBCl-hLF is about 21kb; (2) The target fragment hLF cDNA is much smaller than pBCl, there would be some difficults in ligation; (3) Directional cloning can’t be used because there is only one cloning site on pBCl.We tested different conditions to solve those problems: (1) Optimized the preparation of the component bacteria; (2) Optimized recovering the fragments of DNA from agarose gels; (3) Optimized the ligation system; (4) Optimized the dephosphorylation of plasmid DNA; (5) Tested the temperature of ligation.The results show that: (1) The concentration of the nucleic acid is the most important factor. The better result could be obtained when the concentration of the nucleic acid is higher than 100ng/μl and the molar ratio of plasmid vector to insert DNA fragment was approx. 1:3 in the ligation. (2) The purity of the nucleic acid would affect the effect of the transformation. (3) PEG8000 may be harmful to the component bacteria; it would be discarded from the nucleic acid before transforming. The target clone was obtained when the concentration and the purity of the nucleic acid were adjusted and the PEG8000 was discarded.The experiment explored the conditions of constructing the mammary gland expression vectors using pBCl.

Related Dissertations

  1. Construction and Agroinfiltration Analysis of RNA Inference Vectors Against Potato Virus Y,S435.32
  2. Construction of pBI121-Lyz-GFP Expression Vector and Its Expression in the Callus of Alfalfa,S541.9
  3. The Expression of Heat-labil Enterotoxin LT in Tobacco,Q943.2
  4. Construction of TIMP-3 Gene Eukaryotic Expression Vector and Study on Its Function in Human Prostate Cancer Cell Line,R737.25
  5. Study on Construction of Mammary Gland Specific Expression Vector for Ssβ-gly and Its Transfection of Bovine Fetal Fibroblasts Mediated by Liposome,Q78
  6. Effect of RNA Interference Targeting of PI3K on the Proliferation of Vascular Smooth Muscle Cell and the Intimal Hyperplasia,R654.2
  7. The Construction and Characterization of Human Prourokinase Mutant,R91
  8. Expression of Lumbrokinase Gene in Goat Mammary Gland and Construction of Lumbrokinase Transgenic Mice,Q814
  9. Endosperm-specific Expression Vector Construction and Genetic Transformation of Wheat Avenin-like b Gene,S512.1
  10. Activity Analysis and Construction Recombinant Procaryotic and Adenovirus Vector of IL-24、Proteomics Study of AdIL-24,Q78
  11. Agrobacterium Tumefaciens Mediated Transformation in Lolium Perenne L.,S688.4
  12. New antibacterial peptide design , synthesis and expression of the gene,Q789
  13. Cloning of Phenylalanine Ammonia-lyase from Caragana Korshinkii Kom, Construction of RNAi Vector and Anaylysis of Its Function,S548
  14. Expression and Purification and Activity Analysis of Human β2-Defensin and mRNA Detection in Gingival Epithelium,R78
  15. The Study of the Effect of RNA Interference on HIF-2 in the 786-0 Renal Cancer Cell,R737.11
  16. Genetic Transformation of Carrot (Daucus carota L.) with ETEC Colonization Factor Antigen Gene CS3 and Construction of Virus-based Gene Vector for Expressing LTB-ST Fused Gene,Q789
  17. The Study on the Regulation of Osmotic Pressure and the Ultramicro Structure of Dunaliella Salina, the Cloning and Analysis of Gene Enolase, and the Identification of Transgenic Tobacco,Q785
  18. Construction of Recombinant Adeno-associated Virus Expressing HBV Envelope Major Protein, Large Protein and the Preliminary Study of Their Immunogenicity,R392
  19. Cloning of Key Genes during Osmotic Stress Signal Transduction and Genetic Transformation of DREB1A Gene into Rice,S511
  20. Carrot (Daucus carota) antifreeze protein gene cloning and transformation of tomato (Lycopersicon esculentum),S641.203.5
  21. The Studies on Constructing Expression Vector of Nematode Resistance Gene and Genetic Transformation,S336

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