| Numerous current data show that, besides anti-keratin autoantibody (AK auto Ab), hundreds of natural autoantibody (NAA), comprising a huge and complicated network of autoantibody in human immune system, have been found to react with a large panel of self constituents, such as MHC, RBC, DNA, transferin, keratin, diphosphatidylglycerd, idiotype of IgG, etc. Although its biological roles remain unclear, NAA is generally believed to play a very important role in immune homeostasis, instead of a horror autoxicus.As a member of NAA family, AK auto Ab is a kind of NAA against tonofilament in keratinocyte (KC), thus having drawn more attention from many dermatologists, who have made numerous studies on it. AK auto Ab was first found by Krogh in 1970, and could be produced spontaneously by mouse spleen cells withnot deliberate immunization with the target antigen. In vitro experiments by Hintner showed that IgG AK auto Ab can promote the phagocytosis of intermediate filament aggregates by human monocytes and polymorphonuclear neutrophils. However, the knowledge of its physiology and pathology is still scarce because indirect research means have hindered its further investigations for a long time. Later, we successfully purified and identified AK auto Ab by affinity chromatograph from pooled sera of normal human subjects. It was further found that AK auto Ab can significantly inhibit the proliferation of KC and the production of IL-6 and IL-8 by KC, and induce apoptosis of KC cultured in vitro. Animal experiments with purified AK auto Ab showed that it can promote the recovery from variousdermatitises, such as allergic contact dermatitis and psoriasis, suggesting that AK auto Ab has a therapeutic potential.However, there are many limitations in clinical application of purified AK auto Ab because it came from human blood. Furthermore, purified AK auto Ab are polyclonal antibodies, containing IgG and IgM of different AK auto Ab against keratins of various molecular weights. So it has yet to be decided whether one or few, or all antibodies contribute to the inhibitory effect on human keratinocyte.The two major problems encountered in current investigations into AK auto Ab are as follows: one is to develop an effective product having no side effects, and the other is to elucidate the molecular mechanisms, especially the mechanisms of signal transduction through which AK auto Ab plays its roles. Therefore, development of a more effective and accurate method to solve these problems has become a must.The present study focused on observing the biological effects of genetic-engineering anti-keratin human Fab fragment, preparing a single-chain antibody of a human anti-keratin antibody and investigating the molecular mechanisms of the antibody's effects.The main results are as follows:The first part: Cloning and expression of ScFv of a human anti-keratin antibody and detection of its acrivitiy.1. A single chain Fv of human anti-keratin antibody was constructed from a human anti-keratin Fab fragment selected from a semi-synthetic phage antibody library. Results of DNA sequence analysis showed that nucleotide sequence of VK and VH of ScFv gene was similar to that of VK and VH of Fab gene, suggesting that there was no mutation in the construction of ScFv. The soluble anti-keratin ScFv was found to have a good antigenic specificity as well as the excellent combining activity with target antigen by ELISA. However, the affinity of ScFv was lower than that of Fab.2. Soluble human anti-keratin Fab fragment was successfully expressed in E. coli and efficiently purified by IMAC. Purified Fab fragment has a good antigenic specificity and excellent combining activities with antigens as shown by ELISA and Western blot. This genetic-engineering antibody only react with the keratin of 48 kilo-dolton, but not with keratins of other molecular weight and other antigens, such as pepsin, desmin, ferritin, human IgG, etc.The second part: Influence of human anti-keratin Fab on the proliferation and apoptosis of b... |
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