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Construction and Immunological Study of a DNA Vaccine from a Hybrid Gene of Protective Antigens of Plasmodium falciparum

Author: YaoLang
Tutor: LiYingJie
School: First Military Medical University
Course: Pathogen Biology
Keywords: Plasmodium falciparum DNA vaccine Antigen DNA immunization Immunology Gene
CLC: R382.31
Type: PhD thesis
Year: 2000
Downloads: 97
Quote: 0
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Abstract


OBJECTIVE Malaria, especially caused by Plasmodium falciparum(P.f), is the most important parasitic disease of man. Most of the 300-500 million clinical cases of malaria per year worldwide occur in developing countries, particularly in Africa. An effective vaccine against malaria would represent a major strengthening of control. Research efforts for development of malaria vaccine to reduce severe and complicated malaria and malaria-related mortality have been made for many years. The complexity of the life cycle, the transmission dynamics in different endemic settings, and the spread of resistance to various drugs by the parasite and to insecticides by the vector render control strategies very difficult. DNA vaccines, since the first demonstration of technology a few years ago, have emerged as a promising method of vaccination. Compared with a panel of conventional vaccines, DNA vaccines have been shown both to induce potent cell-mediated immune response and humoral immune response. They also offer many advantages in terms of ease of construction, testing, and production. Immunization with DNA vaccines provides an exciting new alternative approach to developing malaria vaccines. The target gene HGFSP, synthesized in our laboratory, is a multi-epitope gene encoding the epitopes from MSAI, MSA2, CSP and RESA of P.1 and the T cell epitopes from IL- I and tatanus toxin(TT). It has been proved in the previous studies that the rabbit sera immunized with HGFSP-recombinant protein expressed in E. co/i or with I-JGFSP-recombinant vaccinia virus can inhibit the growth of F~f in vitro. The objective of our research work is to construct a malaria DNA vaccine with I-IGFSP gene and an eukaryotic expression vector pcDNA3 and to observe the in vitro expression of HGFSP gene in mammalian cells and the cell-mediated immune response and humoral immune response elicited by the recombinant pc-I-IGFSP DNA in mice. METHODS HGFSP gene was Sub-cloned into the IlindIII and BamHI sites of an eukaryotic expression vector pcDNA3 from a prokaryotic recombinant plasmid pSK-HGFSP to construct the eukaryotic expression recombinant plasmid, pc-HGFSP. The recombinant plasmid was identified by agarose gel electrophoresis, restriction enzyme analysis and enzyme map analysis. The recombinant was then transfected into mammalian cells, HepG2, with the liposome-mediated transfection. The transfected cell clones were selected by G418 co-culture. The positive cell clones were tested by immuno-fluorescenct assay(IFA), SDS-PAGE and Western-blot to identify the immunogenicity. Plasmids of pcDNA3 and pc-HGFSP were prepared in large scale for DNA immunization. C57BL16 mice were divided into three groups: Normal saline(NS) control; pcDNA3 blank plasmid control; and pc-HGFSP experiment group. They were then injected intramuscularly with bupivacaine in the quadriceps muscles of left leg. One day later, the pc-HGFSP plasmid DNA was inoculated at the dosage of 100 i?g in the same site of the left legs of the mice. Control groups were injected with pcDNA3 blank plasmid or normal saline, respectively. Two booster injections were given at three weeks interval(d2 1 and d42). After two and four weeks(d56 and d70) of the last booster injection, the sera and spleens of tested mice were collected for immunological assays. The spleenocytes were used to detect the killing activity of NK cells and Cytotoxic T lymphocytes(CTLs) by LDH assay, the proliferation activity of T lymphocytes by MT

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CLC: > Medicine, health > Basic Medical > Medical Parasitology > Medical protozoa school > Cryptosporidium > Plasmodium
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