Dissertation > Excellent graduate degree dissertation topics show

Construction of the Secretory Recombinant Vector Expressing E2 Glycoproteion of Classical Swine Fever Virus and Immunological Efficacy Study

Author: XuYanZhao
Tutor: ZhangYanMing
School: Northwest University of Science and Technology
Course: Preventive Veterinary Medicine
Keywords: Classical swine fever virus E2 gene Secretory expression Antibody detection
CLC: S852.65
Type: Master's thesis
Year: 2009
Downloads: 78
Quote: 1
Read: Download Dissertation

Abstract


Classical swine fever (Classical swine fever, CSF) is a classical swine fever virus (Classical swine fever virus, CSFV), pigs caused a highly contagious disease, the disease to the world's pig industry has caused tremendous economic losses. In the prevention and control of swine fever vaccine has played a key role, especially in China successfully developed HCLV vaccine to China even in the world of classical swine fever prevention and control has played a positive role. The vaccine is also inadequate, if they can not distinguish the body to produce antibodies induced by vaccination or infection caused by wild virus, and therefore affect the international trade of pigs and pork. Therefore, the development of differential diagnosis of classical swine fever genetically engineered vaccine is imperative. This study constructed expression of E2 gene of CSFV Shimen strain secreted eukaryotic expression vectors and recombinant adenovirus pAd-lgk-E2sh, and in immortalized porcine secretory expression of vascular endothelial cells, the collected protein Quantitative and conduct preliminary tests in mice immunized for the development of genetic engineering subunit vaccine swine fever basis. 1 gene expression CSFV E2 and E2 removal of the transmembrane region of the eukaryotic secretory Construction and Identification. PCR was first used from the plasmid pMD-19T-E2 E2 gene is amplified sequence (E2qc) and removal of the transmembrane region of the E2 gene sequences (E2sh), were cloned into the eukaryotic expression vector pSec secretory -Tag 2 (A), the successful build pSec-Tag-E2qc and pSec-Tag-E2sh. Respectively, through liposome transfection method so that it will build a good positive vectors in immortalized porcine umbilical vein endothelial cells, by SDS-PAGE and Western blot protein expression and activity, the results show that removal transmembrane region of E2 gene sequences of eukaryotic expression vector transfected cells successfully target protein secreted into the medium, and can combine with positive serum CSF. 2 recombinant adenovirus expressing CSFV E2 pAd-lgk-E2 Construction and identification. First using PCR from pSec-Tag-E2sh plasmid lgk-E2 gene was amplified, cloned into the adenovirus shuttle vector pAdTrack-CMV to construct a recombinant adenovirus shuttle plasmid pAdTrack-lgk-E2. With Pme Ⅰ endonuclease pAdTrack-lgk-E2 was linearized plasmid was recovered after digestion products containing the adenoviral backbone vector conversion pAdEasier-1 BJ5183 competent cells of E. coli shuttle vector and matrix manipulation homologous recombination vector, obtain a solution containing lgk-E2 gene pAdTrack-lgk-E2. Followed by endonuclease Pac I linearized pAdTrack-lgk-E2, recovered through liposome-mediated method skeleton vector was transfected into HEK293 cells obtained recombinant adenovirus containing the target gene pAd-lgk-E2. Control was prepared in the same way an empty adenovirus vector pAd-CMV. The recombinant virus produced in 293 cells typical cell lesions and expressing green fluorescent protein. RT-PCR analysis showed that lgk-E2 gene was successfully transcribed infection immortalized porcine umbilical vein endothelial cells, SDS-PAGE and Western blot showed that the target gene was successfully expressed and has good reactogenicity. 3 of CSFV E2 protein expression in mice immunized tests. The collected supernatants containing the proteins with ultra low temperature method of lyophilization preliminary estimate of the E2 protein of about 100μg / mL. The 24 Balb / c mice were randomly divided into six groups, each 4 by intraperitoneal and intramuscular injection three different immunogenic antigen content, and set positive, negative and blank control, the two times after immunization with swine fever virus antibody ELISA test kit detects antibodies. The results show that by 2 mice after immunization against CSFV-specific antibody, antibody titers of up to 1:1 500.

Related Dissertations

  1. Detection Classical Swine Fever Virus and Porcine Circovirus Type 2 of Microarray Technique,S858.28
  2. Serological Supervision and Prevention of Porcine Reproductive and Respiratory Syndrome in Large-scale Swine Farm,S858.28
  3. Study of High Efficient Secretory Expression of Endostatin Via Adenoviral Vector,Q78
  4. Diagnosis on the Classical Swine Ferver Outbreak in One Pig Farm and Construction of Expression Vector for E2 Gene of Classical Swine Fever Virus,S858.28
  5. Adenoviral vector pAd-E2-pIL-2 and pAd-pIL-2 Construction and Immune Effect Study,S852.5
  6. Mutations of E6 and E2 Oncogenes of Human Papillomavirus 16 in Women with Cervical Carcinoma in Qingdao,R737.33
  7. Chicken Gal-2 mature peptide cDNA fragment in Pichia pastoris expression and secretion of antimicrobial activity,S852.4
  8. Analysis of Mutations and Adaptive Evolution of E2 Genes of High-risk Human Papillomavirus (HPV) in the Patients with Cervical Cancer in Sichuan Area of China,R737.33
  9. Construction and Functional Analysis of Recombinant Bacteria for Expression PeaT1 in Bacillus Subtilis,S476
  10. Dissimilar EGFR extracellular domain of recombinant DNA and protein vaccine preparation and anti- tumor effect in mice,R730.5
  11. Generation of a BacMam Virus Expressing the E2 Protein of Classical Swine Fever Virus and Its Immunogenicity in a Mouse Model,S852.65
  12. Molecular Cloning and Sequence Analysis of Envelope Glycoprotein Gene E2 in Isolation Strain from Bovine Viral Diarrhea Virus of Deer,S852.65
  13. Epidemiological Investigation on Classical Swine Fever in Shandong Province,S858.28
  14. Genetic and Variation Analyses on CSFV E0 & E2 Gene in Parts of China and Sequence & Analysis of the Complete Genome of Epidemic Strains,S852.65
  15. Cloning, Expression and Identification of Human Papillomavirus Type-16 E2 Gene,Q78
  16. Study on the E2 Gene Nucleotide Sequences and Pathogenicity of Prevalent Field Strains of Hog Cholera Virus,S852.651
  17. Study on amplification and its optimum reaction conditions of E2 gene of Hog Cholera Virus Guangxi prevalence strain,S852.651
  18. Study on the E2 Gene Nucleotide Sequences of CSFV field strains and Some Characteristics of Its Multiplication in PK15 Cells,S852.65
  19. High level expression of the major antigenic domains on envelope glycoprotein E2 Of classical swine fever virus in E.coli and purification of recombinant products,S852.65
  20. CLONING AND SEQUENCE ANALYSIS OF E2 GENE OF THE EPIDEMIC VIRULENT STRAINS OF CLASSICAL SWINE FEVER VIRUS IN GUANGXI,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