Dissertation > Excellent graduate degree dissertation topics show

The Experimental Study on Multicomponent Fusion Vaccine of Helicobacter Pylori Outer Membrane Protein

Author: GeDi
Tutor: ZouQuanMing
School: Third Military Medical University
Course: Clinical Laboratory Science
Keywords: Helicobacter pylori Outer membrane protein OMP18 Immune protection Mucosal immune
CLC: R392
Type: Master's thesis
Year: 2007
Downloads: 42
Quote: 0
Read: Download Dissertation

Abstract

Purpose: The global prevalence of Helicobacter pylori (H.pylori) more than 50%, it is a major cause of chronic active gastritis and peptic ulcer and intestinal-type gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma closely related. Existing means to eradicate H.pylori mainly combined antibiotics, but because of the universality of drug-resistant strains of bacteria infection continues to increase, the higher the cost, and low patient compliance, limit the efficacy of antimicrobial agents. Therefore, vaccination is simple, low-cost and easy-to-large scale of H.pylori vaccine research is very urgent. In vaccine research, to screening effective immune antigen is the critical link. Most vaccines used as the antigen component is associated with Hp pathogenic virulence factors, such as urea enzyme (urease), heat shock protein (heat shockprotein, Hsp), vacuolating cytotoxin (vacuolating cytotoxin, VacA), cytotoxin-associated antigen (cytotoxinassociate antigen, CagA), neutrophil-activating protein (neutrophil-activating protein, NAP), etc.. Gram-negative bacterial outer membrane protein antigen activity, these outer membrane proteins as antibodies and immune cells to attack the main target, can be mediated by the most direct and effective role in the killing of bacteria, is to determine whether the immune response is protective of human the sexual key factors. The OMP18 and UerB, are Hp outer membrane protein composition, its encoding gene is highly conserved, Hp vaccine immune antigen. But has been so scholars regret each antigen alone immunized animals, the protection rate is not very high, while a variety of antigens combined immunodeficiency in order to achieve good results. Therefore, this study proposed the two kinds of protective antigen gene cloning and expression of multi-subunit of the fusion protein with molecular adjuvant LTB series, different immune responses were observed in mice induced protective effect for screening H.pylori vaccine antigen to lay the foundation. Method: 1, by PCR from the genome of H.pylori clinical isolates 9806 amplification the OMP18 gene fragment and build the prokaryotic expression vector pQE-30 positive recombinants by restriction enzyme digestion and sequencing. Bioinformatics software DNAssist and GenBank database the published of H.pylori OMP18 gene sequence similarity analysis. The recombinant vector containing the gene fragment was transformed into E. coli M15 with the AKTA-explore Purification Instrument purified expression induced by IPTG, the recombinant protein rOMP18. Application Tris-Tricine expression product purified product analysis with the purified rOMP18 immunizing rabbits, and Western blot and immunodiffusion test recombinant protein immunity. 2, overlap extension PCR method, respectively, as a fusion protein front in UreB414 active fragments, in series with OMP18 and mucosal adjuvant LTB and turn the series as a mucosal adjuvant LTB fusion protein front end with OMP18 introduced between the fragments 5 amino acid linker PQDPP connection; fusion gene construct prokaryotic expression vector pET-28a, after restriction analysis and sequencing, the positive recombinants were transformed into E. coli E. coli BL21 (DE3), IPTG-induced expression of recombinant engineered bacteria. Tris-Tricine electrophoresis and immunoblotting identification the fusion protein DNAssist software analysis fusion protein physicochemical characteristics, to grope through the purification conditions, the AKTA-explore purified Miriam purified fusion protein LTB-OMP18 (LO), and UreB414-OMP18-LTB (UOL). The fusion proteins were purified with GM1 ganglioside binding assays. 3, 144 mice were randomly divided into six groups: the PBS the control group, within the single subunit molecular adjuvant fusion protein the group (UreB414-LTB) within dual subunit molecular adjuvant fusion protein (UreB414-OMP18-LTB The) single subunit molecular adjuvant fusion protein (LTB-OMP18). vitro physical mixed group (rOMP18 UreB414-LTB) molecular adjuvant single subunit protein group (OMP18). Respectively 0,7,14,28-day oral immunization dose of 150μg / /. 10 days after the surgery the third immunization, each group of 12 mice were killed and sampled ELISA detection of oral immunization serum specific IgG, IgA, feces, intestinal washing fluid sIgA level. Each group of 10 ~ 8 CFU Hp mouse-adapted strain of the remaining 12 oral and 30 days, take the mouse stomach specimens by Hp culture and specific PCR to determine Hp infection rate, and calculated the rate of oral immunization groups challenge protection . Results: 1 by restriction enzyme digestion and gene sequencing results display of H. pylori OMP18 gene was cloned into pQE-30. OMP18 nucleotide sequence length of 540bp, in GenBank, the homology was 99% amino acid sequence homology is 100%. IPTG induced recombinant engineered bacteria, analyzed by Tris-Tricine recombinant protein molecular weight of about 20KD expression products accounting for 20% of the bacterial protein is expressed as inclusion bodies, and purified by the AKTA-explore 100 protein purification systems, purification Purity> 85% of the target protein was obtained. Purified protein antigen with Freund's adjuvant in rabbits, prepare rabbit to anti rOMP18 specific antibodies, detected by double immunodiffusion, rabbit anti-serum antibody titers of 1:32, Western Blot results also showed that the recombinant protein to be rabbit anti- identify H.pylori serum. Was successfully constructed by restriction enzyme digestion and gene sequencing results show the fusion gene of LTB-OMP18 and UreB414 OMP18-LTB the the fusion gene LO and UOL cloned into the pET28a vector to fusion protein the expression vector pLO and pUOL, DnaStra software Evaluation protein linker has a good flexibility. Two positive recombinant was transformed into E.coli BL21 (DE3), 37 ℃ culture induced by IPTG, the expressed fusion proteins by SDS-PAGE and Western blot analysis showed that the molecular weight of about 31KD and 43kD. UVP scan confirmed that the fusion protein expression by between 25% and 20% of the total protein. Inclusion body identification test confirmed the fusion protein was expressed as inclusion bodies, identified using affinity chromatography purification method. Purity> 85% of the fusion protein obtained after purification. Two fusion proteins purified with GM1 ganglioside occurred binding reaction, experiments confirmed that the recombinant fusion protein of LTB component having a biological activity of binding to GM1 ganglioside, the fusion protein to maintain the natural activity of the LTB mucosal adjuvant activity. 3, ELISA detection after the fusion protein rLO and rUOL immunized mice serum specific IgG, IgA, feces, intestinal washing fluid sIgA level with the PBS group compared increased significantly, a significant difference (p <0.001), and subunit the immunohistochemistry difference significant (p <0.05), compared with the multi-subunit protein physically mixed group, the difference was not significant (p> 0.05). The immune poisoning, the immune protection rate: rLO group and 67% (8/12), rUOL group was 83% (10/12). Statistical analysis showed that the immune protection rate of multi-subunit fusion protein group compared with the PBS group, the difference was highly significant (p <0.001), compared with rOMP18 group, the difference was significant (p <0.05), compared with rOMP18 UreB414-LTB group , the difference was not significant difference (p> 0.05). Conclusion: 1, was successfully cloned the the H.pylori OMP18 gene, has a high degree of homology with the the H.pylori strains gene sequence GenBank published. The purified recombinant protein rOMP18 has good immunogenicity and reactivity, can be used as the H.pylori gene engineering vaccine candidate antigen. 2, successfully constructed, express fusion protein RLO and rUOL, after purification of the fusion protein to maintain the subunit component and adjuvant independent immune response, the fusion between the genes Linker less influence on the dimensional conformation of the fusion protein. 3 mice specific antibody levels and immune protective effect prove to fusion the protein rLO and rUOL has a good safety and immunogenicity of the protective effect.

The Experimental Study on Multicomponent Fusion Vaccine of Helicobacter Pylori Outer Membrane Protein

Abstract

Related Dissertations