Dissertation
Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Immunology

The Influence of the FR-1 in Heavy Chain (VH) of Antibodies on Secretion

Author ZhuLiLi
Tutor LiangMiFang
School Disease Control and Prevention Center
Course Immunology
Keywords immunoglobulin variable domain Site-directed mutagenesis eukaryotic expression
CLC S852.4
Type Master's thesis
Year 2008
Downloads 19
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Polyclonal serum, monoclonal antibody and gene engineering antibody are three different stages of antibody development, Antibody engineering technique provided new insight for the clinical application of antibody by humanizing mouse antibody or developing human antibody directly. The fully human recombinant anti-virus antibodies are compatible with human immune system, with no immunogenicity, no contaminations, and unique specific anti-virus effect, have been proved to be an emerging bio engineering pharmaceuticals. Moreover, it is easy to be scaled up for a continuous production, which lead them to be an ideal substitutions for sera derived immunoglobulin. Heterologous hosts such as bacteria, yeast, insect cells, mammalian cells, filamentous fungi and various species of plant can been used to express and produce antibodies. In terms of the advantages of helping correct protein folding, assembly and post-transcriptional modification, which is closer to human proteins, mammalian cells is used as the main system in the production of the recombinant protein. However, the expression ability and levels are quite various for different antibody genes in mammalian cells, so high-efficient expression in mammalian cells system is considered a bottleneck restricting the production of antibodies. The major factors related to high-efficient expression for antibody genes in mammalian cells, mostly include antibody gene sequence, the integration site of antibody genes on host chromosome, copies of the antibody genes, transcription and translation level, the choice and modification of host cells and balance expression of light chain and heavy chain genes etc. The N-terminal segment (FR-1) of the heavy chain (VH) of antibodies may have a great impact on IgG secretion in Escherichia coli, and a single amino acid change can result in the loss of secretion .In order to explore the influence of FR-1 of the antibodies heavy chain (VH) on efficient expression in mammalian cells, we changed the amino acid composition of FR-1 with site-directed mutagenesis in different engineering antibodies, to demonstrate the impact on antibody secretion.Site-directed mutagenesis of FR-1 was performed in several kind of human recombinant antibodies developed in our laboratory, including the anti-rabies virus antibody, anti-hepatitis A virus antibody, anti-virus antibody, anti-SARS corona virus nucleoprotein antibody, anti-hepatitis B virus antibody and anti-Hantavirus nucleoprotein antibody. Mammalian cell 293-T was then respectively transfected by the mutants to study the changes of antibody secretion.Prior to the site-directed mutagenesis study, we evaluated and analyzed two aspects: the secretion ability of the antibodies in mammals and the antibody genetic information. A total of eight different antibody genes was transfected into 293-T cells for transient expression. The results indicated that the anti-rabies virus antibody existed intracellularly without a secretion, and the other seven antibodies can be secreted .Then 8 antibody gene sequences were compared for analysis in V-base database, which showed that the gene of the anti-rabies virus antibody heavy chain belonged to the VH1 family, one of the two anti-avian flu virus antibodies heavy chain genes and one of the two anti-Hantaan virus nucleoprotein antibodies heavy chain genes belonged to the heavy chain gene VH4 family, and all the others belonged to VH3 family. The analysis results provided major evidence for the following research, which was carried out by site-directed mutagenesis for secretion expression research.All the 8 antibody genes were mutated with site-directed mutagenesis technique to get 12 mutants with different FR-1 in heavy chain, in accordance with the comparison result in the V-base database. Then the 12 mutants were transfected individually into 293-T cell for transient expression. ELISA was used to detect the IgG expression and sectretion in both cells supernatant and cell lysis. It’s shown that both the original 8 antibody genes and the 12 mutants could synthesize complete IgG molecules intracellularly, and the secretion detection showed that after mutation, two of three mutant anti-rabies virus antibodies can be secreted. Among the other 9 mutants from the original seven genes, one has a significantly decrease on secretion ability, and three lost. Our results indicated that the amino acid composition of the FR-1 in heavy chain (VH) of antibodies indeed have a great impact on secretion. For the mechanism of this factor, we used double immunofluorescence analysis to investigate the co-localization of the four anti-rabies virus antibodies with ER, Golgi and lysosome. From this analysis, we found 2 normal secretion antibodies can co-localize both with the endoplasmic reticulum and Golgi, however, the 2 secretion defect can also co-localize with the lysosome, which indicated that the secretion defect maybe due to rapid H chain degradation. Fluorescent analysis was further used to test the antibody functions, which showed the binding capacity of the 2 secretion defect antibody were significantly weaker than normal secretion antibodies. As the function of antibodies is closely related to its structure, the change of the amino acid composition of the FR-1 in heavy chain (VH) may affect the entire molecular conformation, and thus the antibody molecules appear a slightly structure defects, which may influence the Ig correct folding and make it fail to pass quality control check points .We investigated the impact and possible mechanism of change of amino acid composition on FR-1 in heavy chain (VH), on antibody secretion, with the site-directed mutagenesis technique. The results provide significant evidence for the improvement of efficient antibody production in mammalian cells.

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