Dissertation > Excellent graduate degree dissertation topics show

Identifing "Different Genes" of CSFV in SVEC and Extending cDNA Sequences and Inhibiting CSFV Propagation in PK-15 Cells by shRNA

Author: YeGuiSheng
Tutor: ZhangYanMing
School: Northwest University of Science and Technology
Course: Preventive Veterinary Medicine
Keywords: Classical swine fever virus(CSFV) swine vascellum endothelial cells(SVEC) “different genes” small hairpin RNA(shRNA) NS3 gene
CLC: S852.65
Type: PhD thesis
Year: 2007
Downloads: 151
Quote: 0
Read: Download Dissertation


Classical swine fever virus(CSFV) effected specificly pathopoiesis for swine,CSFV could propagate in more swine cells in vitro culture without cytopathic effect(CPE),but had CPE in swine vascellum endothelial cells(SVEC).It’s that one or some protease which one or some“sensitive genes”(they were named with CSFV“sensitive genes”) expressed splited p125 protein to p80 protein after CSFV infected in SVEC possibly.On basis of previous study, we designed small hairpin RNA(shRNA) for four genes of eleven CSFV“sensitive genes”by biology software and synthetized them.We Screened and identified“different genes”by vector transcribing shRNAs in SVEC. The“different genes”sequences by Rapid Amplification of cDNA Ends(RACE) extending provided a basis for researching functions of different genes in PK-15 cells.It was a basis for gene functions and anti-virus by transfecting shRNAs recombinant vectors targeting CSFV NS3 into PK-15 cells.This study obtained following results:1. We designed shRNAs for four“different genes”by biology software and synthetized them.we annealed double-strands and ligated them into RNAi vector and transformed competent cell of E.coli DH5α.We screened and indentified recombinant plasmids, the results indicated that recombinant vectors were constructed successfully.These recombinant plasmids were transfected into SVEC and inoculated CSFV and observed cells pathological changes during different times.The results indicated that SVEC’s appearance and survival rate was better than control cells and other cells by transfecting recombinant vectors targeting for Y8“different gene”.The Y8“different gene”is CSFV“sensitive gene”with SVEC CPE possibly.2. Did CSFV infect PK-15 cells with CPE by“different genes”?cDNA sequcnce of“different gene”was transcribed reversely by RT primers. 5’-uptream unknows sequenc of Y8“different gene”was amplificated by two inverse PCR primers.3’-downstream unknows sequence of Y8“different gene”was amplificated by specific upstream primer and 3’-adaptor primer.Objective genes was ligated into pMD18-T and pMD19-T simple vector and transformed competent cell of E.coli DH5α.We identified recombinant plasmids by restriction analysis and sequence analysis.The results indicated that 5’upstream unknows sequence and 3’downstream unknows sequence of Y8“different gene”were extended by 5’RACE and 3’RACE.3. We analyzed and designed shRNAs for CSFV NS3 gene by biology software and synthetized them.We annealed double-strands and ligated them into pGenesil-1 vector, the constructing recmbinant vectors for pGene-NS3-1,pGene-NS3-2,pGene-NS3-3 and pGene-NS3-Neg negative control were transformed competent cell of E.coli DH5α. Recombinant vectors were transfected into PK-15 cells by liposome and screened positive cells.Cells were collected for Real-Time PCR and ELISA after CSFV were inoculated into cells in 72h.Real-Time PCR results indicated that shRNAs that were transcribed by pGene-NS3-1 and pGene-NS3-2 and pGene-NS3-3 silenced CSFV gene partly;ELISA results indicated that shRNAs inhibitted CSFV propagation partly.

Related Dissertations

  1. SublyticC5b-9 induced Gadd45γ express Thy-1 nephritis lesions of GMCs apoptosis,R692.3
  2. Molecular Diversity of Rice Stripe Virus in Anhui Province and the Functional Identification of NS3 Gene,S432.41
  3. Analyses of HCV Genotypes, NS3 Variation Associated with Drug-resistance and Cytotoxic T Lymphocyte Epitopes in Patients with Chronic HCV Infection,R512.62
  4. Study on the Development of a RT-PCR ELISA Method and the Diagnositic Kit on the CSF,S854.4
  5. Studies of Expression of Human VEGF Gene in Ovarian Cancer Cells with RNA Interference and Gene Therapy with Endostatin,R737.31
  6. Expression of the Truncated E2 Protein of Classical Swine Fever Virus in Escherichi Coli and Preparation of a Monoclonal Antibody Against E2 Protein,S852.65
  7. Down-regulation Human TERT Gene of Telomerase in A2780 Cells of Ovarian Cancer by RNA Interfering Technology,R737.31
  8. Construction of the cDNA Clone and Chimeric Clones of Classical Swine Fever Virus C-strain Vaccine,S852.5
  9. Cloning and Construction of the Prokaryotic Expressing Vector of Partial Gene of Classical Swine Fever Virus,S852.65
  10. Prokaryotic Expression of the Main Antigenic Domains of the E2 Envelope Glycoprotein of Csfv in E.coli,S852.65
  11. Isolation and Culture of Swine Umbilicus Veins Endothelial Cells and Cytopathogencity of Shimen Virulent Strain of CSFV in Endothelial Cells Culture,S852.65
  12. Research on Expression and Antigenicity of Core Antigen Site of E0 Structure Proteins of Classical Swine Fever Virus,S852.65
  13. Generation of a Monoclonal Antibody Specifically Directed Against the Wild-type Classical Swine Fever Virus,S854.43
  14. Establishment and Application of the Methods of Monitoring and Control Against Classical Swine Fever in the Large Scale Pig Farms in Guangxi,S858.28
  15. Development of Chimeric DNA Vaccine Against Classical Swine Fever and TRIF-induced Enhanced Immune Responses of DNA Vaccination,S858.28
  16. Expression of CSFV E2 Gene in PK-15 Cells and Autologous Animal Immunity Test,S852.5
  17. Isolation and Identification of Infectious Bronchitis Virus in Henan and the Full Genome Sequence Analysis of Isolated Strains HN104 and HN091,S852.65
  18. Isolation and Identification of Low Pathogenicity Avian Influenza (H9) Virus in Henan and Study on the Biological Charactistics,S852.65
  19. Isolation, Identification and Molecular Characteristics of Japane Encephalitis Virus in Henan Province,S852.65
  20. Construction and Application of the Integrated Gene Containing Multi-Mimotopes and VP2 of Infectious Bursal Disease Virus,S852.65
  21. Expression of VP2 Gene of Infectious Bursal Disease Virus and Preparation of Monoclonal Antibodies Against the Recombinant VP2 Protein,S852.65

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