Turbot Parvalbumin Preparation And IgE Binding Ability Changes During Oxidation

As fish is used as the raw materials in food industry more and more widely, theincidence of fish allergen increases year by year. On the one hand, we have to performa strict detection of fish allergen. On the other hand, we have to improve the diagnosiseffect of fish allergen. Many countries in the world mainly adopt the method ofenzyme-linked immunosorbent assay to detect fish allergen in the food. Due to theregional differences in fish and the widening international trade in China, it is veryimportant to research the characteristics of fish allergen in food industry and medicalfield.This study is to investigate the turbot. Recently the consumption and cultivation ofturbot was increasing year by year. We extracted and purified the allergen according tothe properties of allergens. The natural allergen was usually instability and hard topurify. So we took molecular cloning to restructure parvalbumin of turbot. Meanwhile,we studied the effect of oxidition on the activity of the allergen in the storage process.The main results are as follows:1. Standardized allergen extracts are necessary for detection and therapeuticpurposes. Parvalbumin of turbot was a typical small molecular protein. And the threesubunits could form aggregates. Twelve existing or modified extraction buffers wereused to extract the protein of turbot. Then we could select a better method based onthe quantity and quality of obtained extracts by SDS-PAGE, immunoblotting andELISA. When dithiothreitol (DTT) was added toTris and Glycine, the concentrationof protein increased by13.46%and the ability of IgG-binding also increased by6.22%. Meanwhile, we used the western blotting to detect the activity of allergen.Results indicate that the protein of16ku,36ku,41ku and46ku which were extractedby Tris and Glycine and DTT elicited a higher reactivity to the three human seratested by immunoblotting. Compared with the extaction without DTT, the proteinwhich were extracted by the solution with DTT elicited the highest concentration anda higher reactivity. The concentration of protein extracted by Tris and Glycine andDTT was4.51mg/mL. And The extract also elicited a higher reactivity to the threehuman sera. So Tris and Glycine and DTT has the potential of replacing most othermethods used for obtaining allergen (and protein) extracts and is therefore recommended. It was necessary that to establish standardized allergen extractsprogram for clinical diagnosis and treatment. This study was aimed at developing anoptimized protocol for the extraction of fish allergens (proteins).2. This study was to investigate the effects of protein oxidition on the content ofcarbonyl and sulfhydryl of turbot proteins after exposured to a free radical generatingsystem. To determine the alterations on protein functionality groups, turbot allergenprotein was oxidatively stressed by incubation at37℃for different intervals of time.We used SDS-PAGE to analysis the alterations of turbot proteins. And the alterationsof the allergenicity of the parvalbumin were investigated by sera from3fish allergicpatients and rabbit anti-turbot parvalbumin polyclonal antibody. Meanwhile, ELISAwas used to evaluate the change of allergenicity of the parvalbumin.The results showed27%proteins were precipitated when exposed to the hydroxylradical oxidation systems for5h. In addition, the contents of carbonyl groups insupernatant and precipitation increased by9.7and2.0times after5h oxidation,respectively. While the contents of sulfhydryl groups in both proteins decreased by13.5%and29.1%, separately. When compared with the control, the electrophoreticpatterns showed that the contents of12ku,26ku and41ku proteins extremelydecreased. However, the contents of36ku protein increased with increasing oxidativetime, which suggests that the aggregates may be formed via non-covalent interactions.IgE Western-blot indicated the immunogenicity of proteins decreased with increasingoxidation time. Meanwhile, ELISA indicated that the immunogenicity of parvalbuminin both proteins decreased by16.7%and31.5%after5h oxidation, respectively.It is concluded that the oxidation alteres the physicochemical properties of turbotproteins and which may lead to the modifications of chemical structure andfunctionalities of the proteins. The chemical changes occurred concomitantly withprotein functionalities. However, oxidative damage can also cause important loss ofquality. So more researches are needed to do next.3. To get the recombinant turbot parvalbumin, and then study the structure andimmunogenicity of it, finally compare with the natural proteins. The proteincomposition was analysed by SDS-PAGE. The results showed that he recombinantprotein was expressed successfully, which molecular weight is about15ku.Meanwhile the recombinant protein can correspond to rabbit anti-turbot parvalbuminantibody. And the immunogenicity of recombinantion protein is85.76%of the natural protein. So we guess that it is the recombinant turbot parvalbumin. We used thedifferential scanning calorimetric method for determining the thermal stability of thepurified recombinant protein. The results indicated the value of endothermic peak is77.73℃. Then the recombination protein has good thermal stability. Therefore insteadof parvalbumin, it can be applied to the detection of fish allergen.