| Since Krogh first proved the existence of antikeratin autoantibody, many researchers have studied extensivly on the natural existence, biology features, physiological and pathological function of this antibody. Up to present, the results indicate that AK auto Ab may take part in many physiological and pathological procedures of skin, and act as an important regulator. However, because of the limitation of research methods, previous knowledge on the physiological and pathological significance of AK auto Ab is rather limited, and mechanism of production of AK auto Ab is unclear. So it is very important to set up an ideal animal model to study AK auto Ab further.Development and application of antibody transgenic mice made it possible to access the above questions. According to the allelic exclusion of antibody gene expression, once a rearranged antibody gene is introduced into the germline of transgenic mice, this gene is expressed ahead of endogenous antibody gene, and endogenous antibody genes cannot rearrange anymore. Thus the transgenic mouse is expected to have a singlekind of B cells bearing specific antigen receptors. So an animal model with higher serum titer of some antibody will be got to investigate this antibodies' biology, and this animal model also can provide enough B cells with unique specificity for further research on the selection/tolerance mechanism of these B cells.Our previous data have set good foundation for the construction of AK auto Ab transgenic mice. We fused B cells from unimmuned mice with myeloma cells, and succeeded in screening 4 antikeratin natural IgM antibodies. Affinity of these monoclonal antikeratin antibodies vary greatly among each other, and their variable region genes are homologous to germline V genes. These hybridoma secreting natural antibodies can not only contribute to our further research about the feature and biology of AK auto Ab, but also provide very fine materials for our research on the production mechaniam of AK auto Ab.In this research, mouse keratin was purified from mouse epidermis, and thiocyanate elution was applied to compare relative affinity of monoclonal AK auto Ab. A high affinity antibody 3B4 were chosen to construct transgenic mice. 1. Construct and expression of IgH and IgL transgeneRT-PCR was applied to clone the VH and VL genes of hybridoma 3B4 that excretes high affinity AK auto Ab. Then antibody transcription promoter, enhancer and constant region fragments were ligated to construct heavy chain transgene plasmid pIC u -V2H7, and light chain transgene plasmid pBSCk-2Vk4. Electroporation was carried out to transfertransgene into myloma cells for expression, and ELISA, RT-PCR, FCMwere used to detect the expressed IgM.2. Construction of heavy chain transgenic mice and genome type analysisLinearized transgene plasmids were microinjected into pronuclei of zygotes from CBAXC57BL/6 mice, and transplanted into oviduct of pseudo-pregnant mice. About 200 zygotes were injected, and 80 mice were born. Genome DNA from mouse tails was extracted and subjected to PCR and Southern blot analysis. There are 12 transgene positive founder mice obtained.3. Phenotype analysis of transgenic miceAnti-IgM/anti-IgD antibodies, anti-IgM/anti-IgMa/anti-IgMb antibodies were used to analyze allelic exclusion of transgenic mice B cells, and it was shown that 50%~80% B cells express transgene encoded IgM. ELISA was used to evaluate serum antikeratin IgM and total IgM of transgenic mice peripheral blood, and it is found that sera antikeratin IgM level of several lines of transgenic mice is significantly higher than littermate control. However, there is no difference in serum total IgM level between transgenic mice and non-transgenic mice.Anti-B220/anti-IgM antibodies were used to analyze number and maturation of central and peripheral B cells in transgenic mice. It is found that number of mature B cells decreased in bone marrow, spleen, peritoneal cavity. B cells in bone marrow of Tg mice were just one half of that of control, and in spleen,...
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