Dissertation > Excellent graduate degree dissertation topics show

DPV VP19c protein expression, intracellular localization and RNA interference inhibits virus replication preliminary study

Author: XiangJun
Tutor: ChengAnChun；WangMingShu
School: Sichuan Agricultural University
Course: Preventive Veterinary Medicine
Keywords: DPV UL38 gene VP19c protein Prokaryotic expression Expression phase Intracellular localization Expressing green fluorescent protein fusion RNA interference Short hairpin RNA
CLC: S852.65
Type: Master's thesis
Year: 2011
Downloads: 54
Quote: 0
Read: Download Dissertation

Abstract


Currently, have yet to see on DPV VP19c protein-depth study reports. In this experiment, the laboratory in NCBI GeneBank registered DPVUL38 gene (accession number EU071041) conducted a bioinformatics analysis, cloning and prokaryotic expression of recombinant protein purification and polyclonal antibody preparation, expression and intracellular localization phase, RNAi target DPV UL38 gene inhibits the replication of the study, the results are as follows: 1.UL38 genes through bioinformatic analysis UL38 gene and its encoded protein structure, functional sites, antigenic and other rare codons conducted a comprehensive bioinformatics analysis showed that DPV UL38 gene contains a conserved domain, with a variety of α herpes virus protein homologous nucleic acid and amino acid sequences with high homology. The product does not contain a signal peptide coding, a transmembrane region, can be localized in the nucleus, and the epitope at the N terminal more relatively large, does not contain multiple consecutive codons. 2.UL38 Cloning, prokaryotic expression and antibody preparation on the basis of the above analysis of the gene cloning and prokaryotic expression system pET-32a (), respectively, expressing a complete ORF encoding ORF coding products and some products analyzed by SDS-PAGE indicate the size of the fusion protein of approximately 66kDa and 45 kDa, Western blot analysis of the expression product can be used with rabbit polyclonal antibody anti-DPV-specific immune response, ELISA analysis showed that the two proteins antigenicity close. Purification of recombinant proteins, and then prepared by immunizing rabbits corresponding polyclonal antibody titer AGP l: 8. Western blot showed that antiserum could specifically react with the virus particles. 3.VP19c phase protein expression and intracellular localization determined by Western blot UL38 gene encodes a protein phase, the earliest discovered after 8h in virus-infected cells to detect the protein, which is a typical late herpes virus protein expression profile is consistent with The. Determined by indirect immunofluorescence test UL38 genes encoding intracellular localization of proteins, found in the first 8h after infection of cells in fluorescence is detected in the cytoplasm, and then gradually near the nucleus, the nucleus concentrated in 30h. In order to determine the positioning of the protein, was constructed expressing green fluorescent protein fusion vector, the plasmid transfected into duck embryo fibroblasts was observed by fluorescence microscopy confirmed that the protein was active in the nucleus, indicating the presence of a nuclear localization signal. 4. RNAi targeting DPV UL38 gene inhibits replication Currently, RNA interference has not been seen by constructing vector replication inhibition DPV reported. This test is based on DPV CHv strain UL38 gene sequences, water birds known gene sequences and short hairpin RNA expression vector for RNA interference target sequence requirements and selection principles, using BLAST tools specifically selected three strains for DPV CHv small interfering RNA sequence, and then sent to the company to build on the Hai Jikai expression vector. The constructed plasmid and fusion interference expressing green fluorescent protein plasmid cotransfection duck embryo fibroblasts observed under a fluorescence microscope which an interference vector showed better inhibitory effect. Then the best interference screening plasmid duck embryo fibroblasts, 6h after inoculation, to determine by observing cytopathic DPV RNA interference on replication in the cells of the case. Experimental results show that the interference vector of the virus in the cell proliferation to some extent.

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. Expression Dynamics of Pheromone Binding Proteins and Expression Influences by Mating and Knockdown of Cryl in Spodoptera Exigua,S433.4
  10. Molecular Cloning, Mrna Expression and Rnai of Nadph-Cytochrome P450 Reductase Gene in Helicoverpa Armigera (Hǘbner),S435.622.3
  11. Isolation and Identification of Infectious Bronchitis Virus and Sequence Analysis of Its S1 Gene and N Gene,S852.65
  12. Isolationand Identification of Porcine Parvovirus and Parts of Its Biological Characteristics,S852.65
  13. Prokaryotic Expression and Purification of Human Beta-Defensin-9,Q78
  14. Molecular Cloning, Prokaryotic Expression and Characterization of a Novel Trypsin Inhibitor in the Frog, Lepidobatrachus Laevis,Q78
  15. Biochemical Characterization and Molecular Modification of a Novel Marine Mud-derived Pyrethroid Hydrolase,X172
  16. Comparative Study on the Host Choice Mechanism of Helicoverpa Armigera (Hübner) and H.assulta (Guenée),S435.622.3
  17. Effects of BMPR-IB Gene Silencing by Small Interfering RNA on Apoptosis of Porcine Pollicular Granulosa Cells and Ecpression of BMP Pathy-Way-Ralted Genes,S828
  18. Inhibition of Myostatin (MSTN) Expresstion in Primary Porcine Fetal Fibroblasts by Lentivector-mediated RNAi,S828
  19. A Preliminary Study on the Regulation of Hepatoma Cell Biological Behavior by MCFP,R735.7
  20. The Expression of Rabbit Bordetella Bronchiseptica PRN Protein and the Study on Its Immunoprotection,S855.12
  21. Cloning and Expression of Fimn Gene of Bordetella Bronchiseptica in Rabbitry and Establishment of Rapid Detection Method,S858.291

CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
© 2012 www.DissertationTopic.Net  Mobile