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A Preliminary Study on Double-targeted DNA Vaccine by Using Bacterial Ghost As A Gene Carrier

Author: JinXiaoPan
Tutor: JiShouPing；TanYingXia
School: PLA Military Academy of Medical Sciences
Course: Biochemistry and Molecular Biology
Keywords: DNA vaccines Antigen-presenting cells Bacteria slough Foot-and-mouth disease virus
CLC: R392
Type: Master's thesis
Year: 2010
Downloads: 92
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Abstract

The DNA vaccine is one kind of a DNA sequence can be used as a vaccine. This sequence from the pathogen, the encoded protein of pathogens. This sequence was cloned into the eukaryotic expression vector and the constructed recombinant plasmid is injected into the host body, and to the foreign gene to be expressed, thereby activating the body's immune system, triggering antibody response, you can achieve the purpose of the elimination of pathogens. DNA vaccine development is called a revolution of vaccine research, play a major role in modern vaccine research. However, the DNA vaccine itself, the level of immune response is very low and often difficult to achieve the intended purpose. Efficient and specific DNA vaccine into the fundamental way to the antigen-presenting cells, and to make it is to improve the activity of DNA vaccine was highly expressed. DNA delivery vector DNA vaccines can be delivered to the APC, to increase the the antigen presenting level and the level of immune response of the vaccine. Bacterial slough (bacterial ghost, BG) using lyase bacterial lysis, and removal of the cell content after the formation of cavities. BG retain the integrity of the bacterial cell wall structure and antigenic properties, can enhance the immune response of the body to foreign antigens, and itself may play a role in the process against pathogens as vaccines. Furthermore, the intima and adventitia of BG periplasm mutually linking a complete structure of the cell membrane, can be used for loading plasmid DNA. BG also host DC identification, phagocytosis, DNA vaccine targeting import DC cells, enhanced the activity of DNA vaccine. Efficiency of antigen-presenting antigen-presenting cells (antigen present cell, APC) is to determine the immunological activity of key factors. Studies have shown that MHC-Ⅱ chaperone invariant chain (invariant chain, Ii) play an important role in antigen presentation pathway. MHC-Ⅱ before peptide binding, antigen binding groove has been the Ii chain of CLIP occupy. Alternative CLIP, with the antigen epitope, will be able to allow the exogenous epitope into MHC Ⅱ targeting the antigen-binding groove, direct the expression of antigen-presenting to effector cells, thus greatly improving the efficiency of antigen presenting, this method called endogenous Targeting. This topic is intended to BG dually targeting DNA vaccine vector preparation: First BG DNA vaccine targeting into dendritic cells (DC), and access to high-level expression; followed by the encoded antigen gene fragment build Ii carrier endogenous targeting the expression of MHC-Ⅱ molecule product was submitted to further improve the antigen-presenting ability and level of immune response. Reported foot-and-mouth disease virus (FMDV) VP1 epitopes sequence targeting observe a validation of this new strategy for vaccine design intended BG loaded double-targeted vaccine import macrophage cell line and the efficiency of the DC, by mice Model dual targeting the enhancing effect of the vaccine on the level of immune response. The main research content and research results are: 1. Preparation and characterization of Escherichia coli ghost. Plasmid was cleaved pHH43 (containing the E-lysis gene cassette) was transformed into E. coli DH5α the lysis gene-E gene expression induced by thermostat, found after 42 ℃ induced about 2.5h, 98.7% of the bacteria occurs cleaved by centrifugation obtained after lysis of the bacteria, the laser confocal microscope confirmed that proves hollow E. coli ghost form bacteria cells without DNA, DNA electrophoresis experiments confirmed prompts we successfully prepared E. coli BG, lay the foundation for the next experiment . 2.BG Loading the nucleic acid fragment, and the plasmid DNA, and transfected the macrophage RAW264.7 BG with CY5-labeled nucleic acid fragment will be successfully prepared by different concentrations mixed, incubation, collected by centrifugation using MitoTracker the BG after staining, flow cytometry detecting loading efficiency found under optimum conditions, are loaded in the 95% of the BG fluorescence-labeled DNA fragment. PI staining the plasmid pdsRED-N1 loaded on this basis, the loading efficiency of up to 91.98%. Will be loaded with the fungus slough transfected macrophages RAW264.7, 48h after transfection using laser scanning confocal microscopy to observe the RAW264.7 cell endocytosis BG and Red gene transfection efficiency of plasmid pDSRed-N1, found in 50 -60% of transfected cells are observed simultaneously in the green fluorescent signal (i.e. MitoTracker labeled BG) and the red fluorescent signal (DsRed encoding a red fluorescent protein). The results suggest that BG can play the role of exogenous targeting plasmid DNA was introduced efficiently macrophages (antigen presenting cells), and to obtain higher expression levels. Endogenous targeting DNA vaccine 3.FMDV building connected to the FMDV structural protein the VP1 of 21-40,141-160 and 200-213 gene fragments with mice invariant chain (mIi), eukaryotic expression vector pDSRed-mIi build the endogenous targeted DNA vaccine pDSRed-mIi/FMDV, and prepared to have a protective effect against various types of FMDV DNA vaccine. Include: from the vector pQE31-mIi, PCR-get mIi sequence built into the eukaryotic expression vector pDSRed-N1 and FMDV virus the VP1 antigen sequences were constructed to pDSRed the-mIi-N1, we have successfully confirmed by sequencing access to containing FMDV fragments that endogenous targeted DNA vaccines. Expression and purification of 4. VP1 antigen FMDV virus VP1 epitope 141-160 is built into the prokaryotic expression vector pGEX-4T-2 in the recombinant transformed into E. coli BL21, antigen expression induced by adding IPTG using affinity chromatography purified target protein, an anti-anti-GST antibody Western-blotting confirmed we obtained protein VP1 (140-160) with GST tag short peptides, experimental results late FMDV antibody The titers detection lay the foundation. The expression levels of cell transfection and animal experiments using the BG loading the pDSRed-mIi/FMDV transfected RAW264.7 cells 24h after transfection extraction of total RNA, reverse transcription PCR method for detection of FMDV in RAW264.7 , found that none of the BG, and bare pDSRed-mIi/FMDV plasmid group is not loaded plasmid FMDV gene fragment was amplified naked plasmid difficult to enter RAW264.7 cells. While the BG loading pDSRed-mIi/FMDV transfection group can be amplified to a FMDV fragment, suggesting that BG is a good DNA vaccine vector, enable exogenous FMDV gene fragment in the macrophage cell line RAW264.7 cells (antigen presenting cells) highly expressed. BG-loaded DNA vaccine to intramuscular injection immunized mice, biweekly immunization once were immunized six times. With GST tag FMDV141-160 antigen by indirect ELISA detecting serum antibody levels. After immunization, the the the BG loading FDMV vaccine immune significantly higher than naked plasmid injection group. Meanwhile, Ii molecules can significantly enhance the antigen-presenting ability, and further enhance the the DNA vaccine original performance higher than the antibody titer of the group of the Ii-FMDV FMDV group BG Ii-FMDV antibody titers than BG FMDV group. The experimental results show that the dual targeting DNA vaccine immunogenicity better than single-targeted vaccine the single targeted vaccine is better than non-targeted vaccines. In addition, we also detected anti-BG antibody titer of mice in animal experiments and found that the low level of the negative control, naked plasmid injection group serum reacted with BG, while in BG group or load plasmids BG group, serum a high degree of reaction of the antibody with BG, when the serum was diluted 80-fold, so that about 80% of the BG can still show a positive signal, indicating that high titers of anti-BG antibody after BG or BG plasmid immunized animal can be produced in vivo These animals have the potential to generate a strong resistance against E. coli. The experiments suggest that BG while as a carrier, which itself there may be used as a vaccine, the corresponding disease immune protective effect. These results initially confirmed using BG as a carrier, or built into the endogenous DNA vaccine targeting vector could increase immunogenicity of the vaccine, while the use of dual targeting vector technique may further raise the level of the immunogenicity of the DNA vaccine. Vaccine prepared by the present method may also be polyvalent, first, if the BG loading two or more kinds of DNA vaccines to produce an immune response, and on a variety of disease; followed if prepared by the selection of specific pathogenic bacteria BG, then in use after vaccination, it can play a role in the DNA vaccine while certain pathogenic bacteria produce immune protection. We believe that, with the dually targeting the improvement of vaccine preparation techniques, the research strategy may play an important role in the new vaccine research in the future.

A Preliminary Study on Double-targeted DNA Vaccine by Using Bacterial Ghost As A Gene Carrier

Abstract

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