Dissertation > Excellent graduate degree dissertation topics show

Expression and Immunoactivity of the Fusion Protien of Swine Influenza Virus Multiple Epitopes to the Heat-Labile Enterotoxin B Subunit

Author: DingChangGen
Tutor: LiuHuiLi；LiuYongJie
School: Nanjing Agricultural College
Course: Preventive Veterinary Medicine
Keywords: swine influenza virus epitopes antigen Escherichia coli Heat-labile Enterotoxin B Subunit immunization potency
CLC: S852.65
Type: Master's thesis
Year: 2008
Downloads: 60
Quote: 0
Read: Download Dissertation

Abstract


Swine influenza (SI), caused by influenza A virus, is an acute respiratory disease of pigs. This viral disease is epidemic all the year, but most in spring and fall. Clinically there were no medicines to cure SIV except for vaccination now. Considering the disadvantages of SI inactivated vaccines, which are univalent or double valent with H1N1 and H3N2 whole virus, people are try to develop new types of SI vaccines. SIV entered organisms through the upper respiratory tract mucous membrane to cause infection, Therefore the mucous membrane immunity vaccination was the best immunity way to prevent SIV infections. Heat-labile enterotoxin (LT) of Escherichia coli has some functions on mucosal immunogenicity and adjuvant.Nucleotide sequences and three diamensions structures of SIV hemagglutination (HA) genes were analysed by means of soft, T and B cell epitopes were screened and connected by adaptor, and conjuncted with B subunit of LT (LTB). Digested with NcoI and HindIII, PCR product of the gene with T and B cell epitopes was cloned into the expression vector pET-30a(+), named pET-30a-ep. PCR product of LTB gene was digested with Hind III and XhoI, and further cloned into the expression vector pET-30a-ep, designated the expression vectors pET-30a-EL1 or pET-30a-EL2 respectively according to having stop codon TAA. The recombinant plasmids were identified by sequencing, and transformed into the BL21 (DE3) and induced to express by 1mM IPTG at 37℃. TheExpression products were identified by SDS-PAGE, and found to be 33KD (EL1) and 38KD (EL2) as expected respectively. The proteins were purified by affinity chromatography on a nikel-agarose resin. The results of Western blotting revealed the recombinant products could react with the antibody to His-tag, indicating that the two recombinant proteins were obtained as expected. The concentrations of recombinant proteins were determined to be 1158.88μg/mL and 1245.90μg/mL by BCA kit, respectively. KM mices of 8 weeks ages were immuninzed by EL1 or EL2 protein through intramuscular inoculation (i.m), subcutaneous injection (s.i), and intranasally (i.n.) , respectively. After third immunization, serum and nasal elution were detected. Serum and mucosal antibody levels were detemined by indirect ELISA, HI antibody level of SIV was detected using hemagglutination inhibition (HI), and cell immunity level was measured by MTT. On the basis of ELISA square matrix titrate experiment, in order to detect EL1 serum, the optimization condition was EL1 coating antigen in 1: 16 (the 62.5ng/hole), the serum in 1:80, EL1 nosal elution coating antigen in 1: 64 (the 15.6ng/hole), nosal elution 1:3. But for EL2 serum samples, coated antigen with the concentration of 1000ng/hole and 1:160 of serum were best. Similarly, for EL2 nosal elution, coating antigen with 1000ng/hole and 1:3 of nosal elution were best. After the mice were immunized for three times, the serum IgG levels maintained at the high level in the EL1 i.m. group and EL1 s.i. group, and the ELISA antibody titer achieved above 1:10000. There were significant differences in serum IgG levels among the EL1 i.m. group, EL1 s.i. group, EL1 i.n. group and PBS group. But there was no significant difference in serum IgG level between EL1 i.n. group and PBS group (P>0.05). In all immunity groups of EL2 protein serum IgG antibody levels were higher than that of PBS group (P<0.01). Moreover, there was significant difference among the different groups of EL2 protein (P<0.01). The mucosal IgA antibody was not detected in EL1 i.n. group during the entire immunity process. But after third immunization, there was significant difference in mucosal IgA level between EL2 i.n. group and the PBS group (0.01<P<0.05). The results of T lymphocyte transformation assay using MTT method indicated, at 20 days after third immunization, there were significant differences between all the immunized group and control group (P<0.01), and T lymphocyte transformation effect in the EL2 i.n. group was strongest. The HI experiment indicated that, HI antibody titers to SIV H3 arrived above 1:256 in the i.m. and s.i. groups of EL1 and EL2 protein and 1:32 in the i.n. groups, but HI titer was only 1:4 in EL1 i.n. group, which was consistent with ELISA result. It suggested the EL1 and EL2 protein had good immunogenicity. Regardless of the EL2 protein by what way immunity mouse, had the good humoral immunity and cellular immunity response, in which i.n. group had also obtained the ideal partial immunity secretion IgA immune body. HI assay examined the anti-SIV HI antibody, which may suppress red blood cells agglutination activeness of SIV, warrantting the organism eliminate the infection virus. The research obtained recombinant SIV antigen which had been possible to stimulate the cellular immunity, the humoral immunity, the partial mucous membrane immune response. This study established a method for developing the epitope vaccine against SIV subgroups coinfection.

Related Dissertations

  1. Establishment of ELISA Based on Recombinant Protein NS1 of Swine Influenza Virus and Nuclear Localization of SIV NS and NP Proteins,S858.28
  2. Expression of Swine Influenza Virus Epitopes Antigen and Its Immonological Activity in Mice,S852.65
  3. Identification of H1N1 and H1N2 Swine Influenza Viruses and Distribution of the H1N2 Virus in Artificial Infected Pigs,S852.65
  4. Resistant gonorrhea LTB-PorB DNA vaccine combined with recombinant protein vaccine immune-enhancing effects of immunization,R392
  5. Cloning and Prokaryotic Expression of Gene NA of Swine Influenza Virus H3N8 and Development of an Indirect Enzyme-Linked Immunosorbent Assay,S852.65
  6. Discussion on the Relationship Between the Swine Influenza Viruses and the Prevalence of Influenza A/h3n2 in Zhejiang Province,S852.65
  7. The Research of the Public Health Events Reports of Chinese Media,G212
  8. Studies on Protective Efficacy of the Recombinant Lactobacillus Plantarum Expressing HA1 Gene of H1N1 Swine Influenza Virus,S858.28
  9. Study of Pathogenicity of H5N1 Influenza Virus Sw/FJ/1/01 and DK/ZJ/52/00,S852.65
  10. Construction and Immunogenicity Evaluation of Recombinant Adenovirus Expressing Hemagglutinin and Nucleoprotein Gene of H3N2 Subtype Swine Influenza Virus,S852.65
  11. Software Systems for Prediction of microRNAs Involved in Swine and Influenza Virus A/H1N1,S858.28
  12. Immune Response of Mice Against Swine Influenza with a Recombinant Pseudorabies Virus Expressing the HA of H3N2 Subtype Swine Influenza Virus,S852.65
  13. Evolution Analysis and the Establishment of Reverse Genetics System of an H5N1 Swine Influenza Virus,S852.65
  14. Cloning, Sequence Analysis and Construction of Eukaryotic Expression Plasmids for NS1 Gene of Swine Influenza Virus (H3N2),S852.65
  15. Studies on Cloning and Expression of Fusion Gene Combining HCV Epitopes Antigen with HBV Core Antigen,Q78
  16. Molecular Evolution of H1N1 Subtype Swine Influenza Viruses Isolated from the Mainland of China,S852.65
  17. Prokaryotic Expression of M1, NS1 and NP Gene of H9N2 Swine Influenza Virus and Development of ELISA Based on Recombinant Protein NP,S852.65
  18. Primary Development of the Genetic Engineering Vaccine Against Foot-and-mouth Disease Virus,S852.5
  19. Studies on Biological Characterization and Gene Characterization of HA、NA、NS for H3N2 Subtype Swine Influenza Viruses,S852.65
  20. Isolation and identification of swine influenza virus and swine influenza serological surveys,S858.28

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