ABSTRACT
In recent years, there has been an increased interest of researchers in developing efficient plant heterologous expression systems of proteins for a wide range of applications. It represents an alternative to the traditional strategy utilizing bacterial, yeast, insect or mammalian cells. New techniques of identification and characterization and effective methods of plant genetic transformation allow the range of recombinant protein products to be expanded. Great expectations are associated with the use of plants as bioreactors for the production of specific proteins of therapeutic interest. This strategy offers a number of advantages, the most important being: the possibility of a significant reduction in production costs, the safety of the products obtained and full eukaryotic post-translational modifications of proteins. A group of proteins of special interest is pharmaceuticals, and a number of successful experiments have confirmed the possibility of obtaining heterogeneous proteins with therapeutic potential: monoclonal antibodies, vaccine antigens, and a variety of cytokines. This work is focused on selected recombinant proteins belonging to those groups expression of which was achieved in plant cells. These proteins may be used in the future for therapy or prevention of viral, bacterial or cancer diseases.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Antigens/biosynthesis , Plant Proteins/biosynthesis , Plants, Genetically Modified/metabolism , Protein Engineering/methods , Recombinant Proteins/biosynthesis , Vaccines, Edible/biosynthesis , Animals , Protein Processing, Post-TranslationalABSTRACT
Expression of vaccine antigens in plants and delivery via ingestion of transgenic plant material has shown promise in numerous pre-clinical animal studies and in a few clinical trials. A number of different viral antigens have been tested, and among the most promising are those that can assemble virus-like particles (VLP), which mimic the form of authentic virions and display neutralizing antibody epitopes. We have extensively studied plant expression, VLP assembly, and immunogenicity of hepatitis B surface antigen (HBsAg) and Norwalk virus capsid protein (NVCP). The HBsAg small protein (S protein) was found by TEM to assemble tubular membrane complexes derived from endoplasmic reticulum in suspension cultured cells of tobacco and soybean, and in potato leaf and tuber tissues. The potato material was immunogenic in mice upon delivery by ingestion. Here we describe the plant expression and immunogenicity of HBsAg middle protein (M protein or pre-S2 + S) which contains additional 55 amino acid pre-S2 region at N-terminus of the S protein. Plant-derived recombinant M protein provoked stronger serum antibody responses against HBsAg than did S protein when injected systemically in mice. We discuss implications for use of fusion proteins for enhanced immunogenicity and mucosal targeting of HBsAg, as well as delivery of heterologous fused antigens. NVCP expressed in plants assembled 38 nm virion-size icosahedral (T = 3) VLP, similar to those produced in insect cells. The VLP stimulated serum IgG and IgA responses in mice and humans when they were delivered by ingestion of fresh potato tuber. Here we show that freeze-drying of transgenic NVCP tomato fruit yielded stable preparations that stimulated excellent IgG and IgA responses against NVCP when fed to mice. However, the predominant VLP form in tomato fruit was the small 23 nm particle also observed in insect cell-derived NVCP.
Subject(s)
Hepatitis B/immunology , Norwalk virus/immunology , Plants, Genetically Modified/immunology , Viral Vaccines/biosynthesis , Viral Vaccines/immunology , Administration, Oral , Animals , Antigens/biosynthesis , Antigens/immunology , Antigens/isolation & purification , Blotting, Western , Centrifugation, Density Gradient , Female , Hepatitis B Surface Antigens/genetics , Hepatitis B Surface Antigens/immunology , Solanum lycopersicum/genetics , Solanum lycopersicum/immunology , Mice , Mice, Inbred BALB C , Plant Extracts/chemistry , Plant Leaves/chemistry , Plasmids/genetics , Rhizobium/immunology , Nicotiana/immunology , Nicotiana/metabolism , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/biosynthesis , Vaccines, Synthetic/immunology , Viral Vaccines/administration & dosageSubject(s)
Antigens/biosynthesis , Antigens/therapeutic use , Communicable Disease Control/methods , Immunity, Mucosal/immunology , Immunization/methods , Phytotherapy/methods , Plant Preparations/therapeutic use , Plants, Genetically Modified/metabolism , Adjuvants, Immunologic/genetics , Adjuvants, Immunologic/metabolism , Adjuvants, Immunologic/therapeutic use , Animals , Antigens/genetics , Chloroplasts/metabolism , Communicable Diseases , Humans , Plant Preparations/metabolism , Plant Proteins/genetics , Plant Proteins/immunology , Plant Proteins/metabolism , Plant Proteins/therapeutic use , Plants, Genetically Modified/virology , Protein Engineering/methods , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/therapeutic use , Viral Proteins/biosynthesis , Viral Proteins/genetics , Viral Proteins/therapeutic useABSTRACT
Latexin, a carboxypeptidase A inhibitor, is expressed in a cell type-specific manner in both central and peripheral nervous systems in the rat. It is used as a molecular marker for the regional specification of the neocortex. In this study, a cDNA was isolated from a human fetal brain cDNA library. The cDNA (LXN) contains an open reading frame encoding 222 amino acids. The comparison between the deduced amino acid sequences of LXN and latexins of rat and mouse revealed high sequence identity (84.2 and 84.7%, respectively). Northern blot analysis showed that LXN was expressed as a transcript of 1.3 kb in 15 out of 16 tissues examined, except in peripheral blood leukocyte. The expression levels were high in heart, prostate, ovary, kidney, pancreas, and colon, moderate or low in other tissues including brain. It is noteworthy that the tissue distribution of human LXN differs greatly to that of its homologue in the model animal, rat latexin. In addition, the LXN gene contains at least 6 exons and spans 5.9 kb according to the genomic sequence of the clone RP11-79M21 and the gap sequence cloned in this paper. LXN was assigned to 3q25-q26.2 according to the position of the marker SHGC-35682 found adjacent to LXN gene.
Subject(s)
Antigens/biosynthesis , Antigens/genetics , Carboxypeptidases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Tumor Suppressor Proteins/pharmacology , Amino Acid Sequence , Amino Acids/chemistry , Animals , Base Sequence , Blotting, Northern , Carboxypeptidases A , Chromosome Mapping , Chromosomes, Human, Pair 3 , Cloning, Molecular , DNA, Complementary/metabolism , Exons , Gene Library , Humans , Introns , Models, Genetic , Molecular Sequence Data , Nerve Tissue Proteins , Neurons/metabolism , Open Reading Frames , Rats , Sequence Homology, Amino Acid , Tissue DistributionABSTRACT
Rheumatoid arthritis (RA) is characterized by recruitment of leukocytes from the vasculature into inflamed synovial tissue (ST) and synovial fluid (SF), which depends, in part, upon the continued maintenance of chemotactic stimuli. RANTES is a potent chemoattractant for leukocytes including monocytes and CD45RO+ memory T lymphocytes. The aim of this study was to determine the production, the source, and the function of antigenic RANTES in arthritis. We detected antigenic RANTES in SFs from RA and OA patients (100 +/- 22.7 and 72 +/- 30.7 pg/ml, respectively). CM from RA ST fibroblasts stimulated with interleukin-1beta or tumor necrosis factor-alpha contained significantly more antigenic RANTES than unstimulated CM (452 +/- 181.6 and 581 +/- 200.2 pg/ml, respectively, versus 12 +/- 4.4 pg/ml, P < 0.05). PHA-stimulated RA SF mononuclear cells secreted 5- to 15-fold more antigenic RANTES than did nonstimulated mononuclear cells, while LPS induced secretion up to 4-fold. We immunolocalized antigenic RANTES to sublining macrophages (28 +/- 3.7 and 8 +/- 2.0% immunopositive cells), perivascular macrophages (56 +/- 6.9 and 19 +/- 3.4%), and synovial lining cells (37 +/- 5.8 and 60 +/- 10.4%) in RA and OA tissue, respectively. Anti-RANTES neutralized 20.2 +/- 1.3% of the RA SF chemotactic activity for normal peripheral blood monocytes (P < 0.05). These results demonstrate antigenic RANTES in RA and OA ST and SF and identify RANTES as a chemoattractant for monocytes in the RA joint.