Dissertation > Excellent graduate degree dissertation topics show

Construction of the Transfecting Plasmid with the P24 Gene of Toxoplasma Gondii Knocked out and Preliminary Research of Establishment Method on Knock-out Strain

Author: TanZuo
Tutor: ShuHengPing
School: Central South University
Course: Microbiology
Keywords: Toxoplasma gondii electroporation gene knock-out mammalian cell
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 235
Quote: 0
Read: Download Dissertation

Abstract


Objective:To establish TgP24 gene-knockout strains,and Exploration on the application conditions of screening.Tg P24 gene knockout strains.Method:(1)Construction of the targeting plasmid pGB/p5-p3 for Toxoplasma gondii P24 gene(TgP24)knock out.Accoding to the genomic sequence of TgP24,four oligonudeotides primers(P1 to P4)were designed and synthesized to amplify the 5’end 2.5 kb fragment of untramlated region(UTR)and the 3’end 2.89 kb frarment of UTR of TgP24.The PCR were subdoned into pCR2.1-TOPO TA plasmid to generate the recombinant plasmid P24-5’-UTR/ TA and P24-3’UTR/TA respectively.The purified fragment of P24-5’-UTR from the digestion of P24-5’-UTR/TA with KpnⅠand BglⅡwas inserted into the KpnⅠand BglⅡsites of plasmid GRA2/Ble to generate plasmid GRA2/Ble-P24-5’UTR(designated pGB-P5);The purified P24-3’-UTR fragment from the digestion of plasmid P24-3’-UTR/TA with BamH-Ⅰand NotⅠwas inserted into the BamHⅠand NotⅠsites of plasmid pGB-P5 to generate the targeting plasmid pGB-P5/P24-3’ UTR(pGB/P5-P3).(2)The condition establishment of TgP24 knock-out strain in vitro screening.Use the millipore filter(aperture 5.0um)filter and purify RH tachyzoites,Observe relatively the impact on plasmid transfected strains of electroporation parameters,such as electroporation transfection buffer, voltage,capacitance,pulse frequency,the size of electric shock Cup The impact of pest.Observe relatively the impact on the transfected strains of different host cells(HFF,3T3 and L929)in vitro on by drug screening;Observe relatively the impact on the transfected strains screened separatly in cell and combine it with low temperature treatment ecto-cells by drug screening(selectively cultured ten years in cells.collected the strains,4℃in extracellular choose five days, retropositioned to the cells cultured in the phleomycin selective medium for seven days).(3)The establishment、identification and analysis of toxoplasma P24 gene knock-out strains.Apply the optimized conditions of electroporation method for transfering RH tachyzoites;use the best conditions for screening transferred strains,then serial subcultivation,High-power microscope observe and count the formation of parasitophorous vacuole(PV),large collection and purify Toxoplasma strains,utend RT-PCR and Western-blot two methods analysis about gene and its protein expression of Toxoplasma P24 gene knock-out strains,and research on the toxicity of gene knock-out strains.Results:(1)Experimental results showed that the targeting plasmid pGB/P5-P3 was constructed by inserting 2.5 kb and 2.89 kb DNA fragment corresponding to the 5’end and the 3’end UTR of the TgP24 gene into the plasmid GRA2/Ble.The fragment P24-5’UTR was inserted into the poly linker site Kpn-Ⅰ/BglⅡupstream of the phleornycin resistance gene(ble),and the P24-3’UTR frament inserted into BamHⅠ/NotⅠsite down-stream of the same gene.(2)Microscopic examination revealed the purity of RH tachyzoites can reach 95 percent,the recovery rate of filtered strains also can reach around 70-80 percent.Optimized conditions of electroporation has improved the survival rate of RH tachyzoites(P<0.05);We relatively observated,the cells will be used within cells,use the method of drug screening on strains in cells and low-temperature treatment ecto-cells will be more thorough;Use L929 fibroblast cell as the host cell for screening,it is easy to form a single Layer,large-volume individual,many passage frequency and slow growth,and the most strong resistant to drug.(3)High-power microscope observe and count,after about 12hr,the number of the transfected strains PV are 8/hp(per high power microscope),significantly lower than the number of the RH tachyzoites PV are 19/hp(P<0.05);Then large collection and purification of P24 gene knock-out strains,RT-PCR,Western-blot showed that the gene and protein expression product of strains screened in L929 cells were significantly lower the groups of strains screened in HFF ang NIH-3T3; animal toxicity test results showed that mean survival time of mice attacked strains screened in L929 cells is longest.Conclusion:(1)The construction of P24 gene knock-out plasmid pGB/P5-P3 was successfully achieved,and amplified fragment at both ends of the target gene is longer,it will benefit to improve the homologous recombination efficiency.(2)Ascertain the application conditions of Toxoplasma electroporation technology,and the best screening conditions of TgP24 knockout plasmid transfected into RH strains cultured in different mammalian cell lines(use drug screening in cells and low-temperature treatment ecto-cells,drug selective concentration within 5.0μg/ml-7.5μg/ ml phleomycin,and L929 fibroblast cell have long screening time and good screening effect as selective host cell)(3)Preliminary obtaining the toxoplasma P24 gene knock-out strains.It has laid a favourable foundation for further research on the biological characteristics of TgP24 knock-out strains.

Related Dissertations

  1. Electroporation Assisted Surface-enhanced Raman Spectroscopy for Living Cells,R318.51
  2. Functional Analysis of tal (transcription activator-like) Genes of Xanthomonas Oryzae,S435.11
  3. Improvement in Electroporation Transformation Efficiency of Gene for Bacillus Subtilis with Trehalose,Q78
  4. Functional Analysis of Tal(Transcription Activator-like) Genes of Xanthomonas Oryzae,S435.111.4
  5. Study on Mechanism of the Th17/Treg Imbalance in Toxoplasma Gondii Infected Pregnant Mice,R714.2
  6. Construction of Dnavaccine about Candida Albicans Hsp90 and Studies on Its Immunological Activity,R392
  7. Structure Analysis of the Virulence-related Plasmid from Vibrio Harveyi VIB 645 and Preliminary Study of the PLD Family Protein from Edwardsiella Tarda,S941
  8. PEF AT2R mediated gene expression in the vascular localized impact on neointimal,R541.4
  9. Construction of Recombinant Plasmid Containing Toxoplasma Gondii HSP70 Gene and Cellular Immunologic Response of Mice Induce by Recombinant Plasmid,R38
  10. Bioinformatics Analysis, Cloning, Expression of PGAM2 Gene of Toxoplasma Gondii and Immunoprotection of rTgPGAM2,R38
  11. Functional Study on the Genes in Biosynthetic Pathway of an Aromatic Polyketide Antibiotic Medermycin,Q93
  12. Effect on the Rat MHC IB and NK Receptor at Maternal-fetal Interface Infected by Toxoplasma Gondii During Early Pregnancy,R714.2
  13. The Role of Toxoplasma Gondii Excreted-secreted Antigens in Some Subpopulations of Murine Immune Cells,R392
  14. The Study of Inonotus Obliquus Polysaccharide on Pathology Change for Mice Infected with Toxoplasma Gondii,S858.91
  15. The Application of Novel Antigen Delivery Vector on Vaccine Against Toxoplasmosis,S855.9
  16. Thermophilic Bacillus Genetic Transformation of ethanol technology research,Q78
  17. Kallistatin electroporation -mediated gene expression in mouse models of IBD preventive treatment,S858.91
  18. Construction of Human Bladder Carcinoma-specific Full-length Antibody Display Library and Construction of the Dual-expression Vector pDGB4,R737.14
  19. The Experimental Study of Silencing Livin Gene by Transfecting shRNA Expression Vector into Pancreatic Cancer Sw1990 Cells with Electroporation,R735.9
  20. Primary Human Lymphocytes Transduced with HLA-A2 Restricted、ny-eso-1 Antigen-specific TCR Genes for Use in Adoptive Immunotherapy for Hepatocellular Carcinoma,R735.7
  21. Preparation of Anti-he4 Monoclonal Antibodies for Detection of Ovarian Cancer,R737.31

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