Dissertation > Excellent graduate degree dissertation topics show

Construction of HBD-3 Gene Breast-Specific Expression Vector and Eukaryotic Expression

Author: PengWei
Tutor: ZhangYong
School: Northwest University of Science and Technology
Course: Clinical Veterinary Medicine
Keywords: humanβ-defensin-3 Holsteinβ-casein EGFP reporter gene donor cells nuclear transfer
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 38
Quote: 0
Read: Download Dissertation

Abstract


The mammary glands of transgenic animals are an ideal site for producing complex bioactive proteins. This technology overcomes the limitations of conventional and recombinant production systems for recombinant proteins. HBD-3 protein is lower in normal human tissue, and difficult to separate or purify, though the extraction of available amount of HBD-3 protein is very limited. However, the cost of chemical synthesis is too costly, so obtain a large number of antimicrobial peptides HBD-3 protein by genetic engineering techniques as a preferred way. Although the antimicrobial activity and biochemical characteristics of recombinant HBD-3 in which the prokaryotic expressed is no difference to natural HBD-3. When we insert the eukaryotic genes into prokaryotic cells, that caused the antimicrobial peptide activity and expression decreased because of the preference of codon usage, Also may be due to expression HBD-3 protein in the form of inclusion bodies in E. coli, purified protein needs to be further to the renaturation with biological activity.Therefore, this study Holstein mammary epithelial cells using eukaryotic expression systems.To establish humanβ-defensin-3 gene transgenic cell lines as competent donor cells for the production of transgenic animals using somatic cell nuclear transfer (SCNT). Firstly, we obtained humanβ-defensin-3 by RT-PCR from human placenta, The amplified DNA fragments were purified from agrose gel, subsequently cloned into pMD-19 vector, and then transformed into E.coli DH5α. The positive clones were named pMhD, which were identified by restriction endonuclease digestion and sequencing analysis. and subsequently inserted the fragment hBD into the corresponding site of the plasmid pBCP. HBD-3 gene was cloned into middle of 5’ end regulatory element and 3’ end regulatory element of the bovine beta-casein (β-casein, CSN2)promoter, the recombinant plasmid pBCD was constructed. We obtained bcd fragment which was 3.0 kb with SacI/ SacII digestion, then was cloned directionally into expression vector pEGFP-C1. The mammary gland-specific expression vector pEBCD was constructed, which comprised of HBD-3 gene gene driven by CSN2 regulator sequence and EGFP reporter gene.The mammary-specific expression vector pEBCD was introduced into the Holstein Fetal fibroblast cells using liposome. After 24-48 h, the expression of report gene EGFP was observed under fluoroscope. Then the transfected Holstein Fetal fibroblast cells were selected by G418 for 3-4 weeks. We identified G418 resistant transfectants by PCR, RT-PCR and EGFP detection. Our results indicated that humanβ-defensin-3 gene stably was integrated into the open region of the chromatin in G418 resistant fibroblast cells. Meanwhile we induced by insulin, hydrocortisone and prolactin to express HBD-3 protein. The recombinant HBD-3 protein from culture supernatant of transgenic mammary epithelial cells was harvested after 48 h, The results of Western blotting ,showed that the transfected cells could expressed recombinant HBD-3 protein, whose MW was 5 ku . Further confirmed that HBD-3 gene can be integrated into the host cell’s genome, making it able toβ-casein protein for the promoter to be highly expressed in favor of the purified protein, which is through the use of animal mammary gland bioreactor for the production of medicinal lay the foundation for protein. This study may provide competent transgenic donor cells for the production of transgenic animals by SCNT and improve the efficiency of transgenic cloning.

Related Dissertations

  1. Vitrification of Porcine Early Stage Embryos Reconstructed by Somatic Cell Nuclear Transfer,S828
  2. Detection of Spindle Morphology and Associated Proteins of Human Somatic Cell Nuclear Transfer Embryos,R321
  3. Production of Bovine Reconstructed Embryos by Whole-cell Intracytoplasmic Injection,Q813
  4. The Techonoly Research on the Elite Boars Produced by Somatic Cell Nuclear Transfer,S828
  5. Effects of Nuclear Maturation Inhibitors on the Maturation and Development of Porcine Oocytes in Vitro,S828
  6. Swines from Gene Knockout with Genes MSTN and GGTA1 by Homologous Recombination Vector and Somatic Cell Nuclear Transfer,S828
  7. Optimization of Platform for Bovine Somatic Cell Nuclear Transfer,S823
  8. Transgenic Sheep of Skeletal Muscle Specific Expression IGF-1,Q78
  9. TSA Increased the Rate of the Blastocysts and Estrus Synchronization Treatment on the Embryo Transferabstract,Q813
  10. Effects of bFGF on the Nuclear Translocation of Angiogenin and Cell Proliferation on A375 Cell Lines,R730.2
  11. Application on the Micromanipulation Enucleation is Assisted Uorescence Methods of Oocyte for Somatic Cell Cloned in Yanbian Yellow Cattle,Q813
  12. Effect of 5-Aza-dC on Development and DNA Methylaton of Bovine Nuclear Transfer Embryos,S823
  13. Construction of hBD3 Expression Vector with Mammary Speciality and Production of Boving Cloned Embryos Transfected with hBD3 Gene,Q789
  14. Effect of Oocytes Source and in Vitro Culture Systrem on the Rate of Nuclear Transfer in Goat,S827
  15. The same kind of sheep and sheep - goats heterogeneous nuclear transfer,S826
  16. Study of hTERT Transfected BFF for SCNT Donor,Q813
  17. Study on Black Pig of Putian by Somatic Cell Nuclear Transfer,S828
  18. Pig Somatic Cell Nuclear Transfer Transgenic Technology Research,S828
  19. Effect of TSA Concentration and Treatment Period on the in Vitro Development and Transgenic eGFP Expression of Pig Somatic Cell Nuclear Transfer Embryos,Q813
  20. Production of Somatic Cell Cloned Bovine Embryos by Whole Cell Intracytopalsmic Injection and Quality Identification,S823

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