Preparation Of High Purity Ovotransferrin From Soft-Shelled Turtle Eggs,Study On Its Immunomodulatory Function And Application

Soft-shelled turtle egg ovotransferrin(TOVT)is the main protein in Soft-shelled turtle white,which has various biological activities such as immunomodulatory effects.However,compared with chicken egg ovotransferrin(COVT)and bovine lactoferrin(BLTF),the differences among different transferrin need to be further studied.In this study,TOVT with high purity was prepared from soft-shelled turtle egg white.The structural properties,linear epitopes and immune function of TOVT were studied,and the immunoregulatory functions of different transferrin and their effects on intestinal flora were explored.Finally,TOVT was used to develop an oral liquid with immune regulatory functions.The main research results are as follows:1.TOVT was isolated and purified from Soft-shelled turtle egg white by DEAE Sepharose FF.The purity was greater than 95% as determined by SDS-PAGE,and the molecular weight was 76024.26 Da by mass spectrometry.The structural differences of TOVT,COVT and BLTF were compared by circular binary spectrum and Fourier infrared spectrum.The results show that TOVT,COVT and BLTF are mainly proteins with β-fold and irregular coil conformation,and the total proportion of α-helix and β-fold structure of the three kinds of transferrin is close.The results showed that the proportion of TOVT α-helix was significantly lower than that of COVT and BLTF(P < 0.05),and the proportion of TOVT β-angle was significantly lower than that of BLTF(P < 0.05).All three types of transferrin possess the typical characteristic peaks of carbohydrate and protein compounds,which are all glycosylated proteins.2.Through bioinformatics technology,the homology relationship between TOVT,COVT and BLTF was studied,and the 3D structure of TOVT was established by using COVT(PDB＿ID 2D3I)as the structure template,and its sensitization and B-cell linear epitopes were predicted.The results showed that the homology between TOVT and COVT and BLTF was 58.04% and 51.78%,respectively.The homology between OVT and BLTF was close to that between COVT and BLTF.Romachandram Plot,VERIFY 3D and ERRAT value evaluation of TOVT 3D structure are all in line with the standard.3.According to Allergen Online allergenic prediction,TOVT was found to have the allergenic fragment Gald 3,which may be potentially allergenic.ABCpred,IEDB and DNAsta bioinformatics software were used to predict 15 possible linear epitapes of TOVT,which are as follows: AA152-156,AA219-228,AA493-496,AA420-428,AA575-583,AA563-568,AA420-424,AA295-297,AA575-583,AA175-177,AA420-424,AA447-452,AA282-288,AA575-581,AA352-357.4.Establishing a mouse model of immunosuppression by CTX,which investigated the effect of TOVT,COVT and BLTF on intestinal immune regulation in immunosuppressed mice.The results showed that different treatment groups had alleviating effects on weight loss,intestinal injury,spleen enlargement and thymus atrophy of mice.The improvement effect of low dose TOVT on spleen enlargement was more significant than that of medium dose and high dose.The effect of TOVT and BLTF on the decrease of thymus index caused by CTX was more obvious than that of COVT.Different treatment groups can regulate serum and intestinal cytokines(TNF-α,IFN-γ,IL-4,IL-10)to some extent,and improve the imbalance of Th1/Th2.The regulation effect of low dose of TOVT is stronger than that of medium and high dose of TOVT.The Th1/Th2 regulation ability of TOVT and BLTF is better than that of COVT.The results showed that the three different sources of transferrin had immunomodulatory functions,and the regulatory capacity of TOVT and BLTF was stronger than COVT.5.Establishing a mouse model of immunosuppression by CTX,which was to explore the regulation of TOVT,COVT and BLTF on the structural disorder of intestinal flora caused by CTX.The results showed that different treatment groups promoted inhibited the relative abundance of Firmicutes,Lachnospiraceae and Lachnospiraceae＿NK4A136＿group.The expression level of relative abundance of Firmicutes,Lachnospiraceae and Lachnospiraceae＿NK4A136＿group was inhibited.There was no dose-dependent relationship between different doses of TOVT.TOVT and BLTF had stronger regulatory ability to promote the growth of beneficial bacteria and inhibit the growth of harmful bacteria than COVT.The comprehensive results showed that TOVT,COVT and BLTF could improve the structure of intestinal flora in immunosuppressed mice.6.A TOVT oral liquid with immune regulatory function was developed,and CTX-induced immunosuppressed mice were taken as objects to verify the immune regulatory function of the oral liquid.The results show that: TOVT oral liquid can improve the weight loss,spleen enlargement and thymus atrophy of mice(P < 0.05),repair the intestinal mucosal damage caused by CTX,and significantly increase the expression of cytokines TNF-α,IIL-4,IL-10,immuneglobulin(Ig A)in serum and IL-4 and IFN-γ in intestinal tissue.Repair the Th1 and Th2 imbalance caused by CTX,has an immunomodulatory effect.TOVT oral liquid can increase the relative abundance of beneficial bacteria in Firmicutes,Lachnospiraceae and Lachnospiraceae＿NK4A136＿group.Reduce the relative abundance of Campylobacterota,Marinifilaceae,Helicobacter,Mucispirillum,Proteobacteria and Helicobacteraceae.