| Summary: | 碩士 === 國立中興大學 === 生物化學研究所 === 94 === Warfarin, a kind of anti-coagulants, has been widely used in clinical medicine. The major function of warfarin is to destroy oxidation-reduction system of vitamin K, therefore the activity of coagulation factors being activated by vitamin K will decrease sharply by warfarin.Warfarin is then employed to cure the diseases such as thrombi, apoplectic and embolic gangrene. However, if patients take a massive overdose of warfarin will cause side effects like hematuresis, bleeding or other serious damage. Moreover, those who take warfarin may probably have the difficulty of coagulating when they are under surgery and will face the dangerous situation of incessant blood stream. To avoid the medicine risk like this, albeit most doctors take great quantities of vitamin K to neutralize toxicity, the result is not correspondent with the expectation. There will be a remedy to apply specific antibody to neutralize the toxicity of warfarin. It can not only scan acutely the exist of warfarin, but also control effectively the side effects of overdose.
Concerning the reproduction and non-specific interaction of polyclonal antibody , the yield and the factors of immunological tolerance of monoclonal antibody. Our main purpose of this experiment is to adopt the phage display system to produce monoclonal single chain fragment of variable region in order to neutralize warfarin. First of all, we constructed Antibody phage display library of warfarin, after repeated affinity selection, we selected the bacteriophage clones which have stronger affinity to obtain 59D4 .
The DNA segment of 59D4 is then thansferred to an appropriate host cell to proceed an protein overexpression. However, with the process of overexpression, the target protein presented as non-activity inclusion body. After trying different inductions and expression methods, we still could not get a great quantities of soluble protein. With the ways of denature and refolding, we acquired soluble scFv. By testing and assuring, we proved that the soluble proteins are equipped with the activity to bind antigen. After that, we will analyze the dissociation constant between warfarin with scFv 59D4 by Biacore experiment and try to apply for clinical trial. We truly infer this antibody can be employed in monitoring the content of warfarin inside human’s blood and proceed to neutralize and to decrease toxicity of warfarin in human.
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