Preparation Of Fab Fragment Of Immunoglobulin Of Egg Yolk And Its Anti-inflammatory Activity

Inflammation is an adaptive immune response to injection and injury.Controlled inflammatory response is beneficial to the body,while disregulated chronic inflammatory response can cause a series of chronic diseases?cancer,atherosclerosis,rheumatoid arthritis,etc.?.Immunoglobulin G?Ig G?,obtained from the blood of healthy human,has shown significant anti-inflammatory effects in various model systems,such as lipopolysaccharide/phytohemagglutinin-induced human whole blood cells.Immunoglobulin of egg yolk?IgY?is similar to Ig G in structure and function,however,IgY has many advantages than Ig G,such as high yield,high affinity for mammalian antigens and not reacting with rheumatoid factors.Its crude IgY extract has been widely used in immune activity and anti-inflammatory activity.However,the IgY Fc fragment cannot activate the corresponding immune regulation mechanism of the mammalian system,which could increase the risk of IgY as a mammalian allergen.At present,methods for separating and purifying Fab and Fc fragments are often combined with multiple chromatography columns.This method is expensive,complicated and time-consuming.Therefore,in this paper,a high-efficiency,environmentally friendly purification methold was developed to obtain high-purity Fab fragments.Then the difference of conformation and physical properties between Fab fragment and IgY were compared.Finally,the anti-inflammatory effect of Fab fragment and IgY in LPS-induced RAW264.5 inflammation model was further studied.The results are as following:Firstly,in order to obtain largest amount of Fab and Fc fragments,the optimal conditions for enzymatic hydrolysis of IgY under papain and pepsin was studied.The hydrolysis degree of IgY could reach 96.8%under conditions:the mass ratio of IgY to papain:25,cysteine concentration:100 m M,and digesting time:5 h.Anti-Fab and anti-Fc immunoblot analysis showed that IgY was mainly hydrolyzed into double-chanins Fab?45 k Da?fragments,single-chain Fab fragments?20 k Da?,and double-chanins Fc fragments?60 k Da?and single-chain Fc fragments?30 k Da?.When the mass ratio of IgY:pepsin was 40 and the digesting time was 1 h,IgY could be hydrolyzed into Fab fragments completely.What`s more,after loading the pepsin digesting on the DEAE FF column,all the Fab could be eluted out in one peak,and the purity of the Fab fragment protein is 97.8%,and the yield was 5.3/100 mg IgY.In summary,the way of pepsin digesting only produced Fab fragment with high purity and yield.While the way of papain digesting can obtain two active fragments,Fab and Fc,and it need further research to obtained high purity Fab and Fc fragment from digestion.Secondly,this study aimed to establish a green,efficient separation and purification method for the preparation of Fab and Fc fragments by papain digestion.IgY papain digestion was firstly treated with 45%saturated ammonium sulfate to remove small molecular weight peptides located near 20 k Da.Subsequently,the mixed solution containing Fab and Fc fragments was loaded onto a DEAE-Sepharose ion exchange column,and the Fab fragments were firstly washed out with 10 m M Tris-HCl buffer?p H 7.6?.Then,the Fc fragments which were bound to sepharose ligand can be elute out lately with 10 m MTris-HCl buffer?p H 7.6?containing 0.21 M Na Cl.The analysis of WB and MS showed that this method yields high-purity Fab and Fc fragments.SDS-PAGE analysis showed that the purity of the two fragments was 88.7%and 90.1%,respectively.The result suggested that this method is simple,easy,and can separate Fab and Fc fragments stimutaneuly.Third,the protein conformation and physicochemical properties of IgY and Fab fragments were stuied.CD spectrum analysis showed that,compared to intact IgY,the?-sheet of the Fab fragment protein increased and the random coil decreased,which indicated that the Fab fragment structure was more stable.FTIR spectroscopy showed that the intensity of the glycans vibration peak increased at 1396 cm^-1 and 1099 cm^-1,indicating that the relative glycan content of Fab fragment increased after being treated with digestion.This phenomenon may be cause by the exposour of glycans which should have been enburied in the hydrophobic environment.In addition,the exposure of the glycans would conversely make the hydrophobic group embedded inside protein,which finally resulted in inceasing the endogenous fluorescence intensity and decreasing ultraviolet absorption intensity.Therefore,the surface hydrophobicity of the Fab fragment is lower than intact IgY.Finally,the physical properties of the Fab fragment and IgY were analyzed,and it was found that the Fab fragment posseses characteristics of lower molecular weight?44 k Da?,lower particle size distribution?4.152±0.473 nm?,and more electric charge on the protein surface?Zeta,-20m V?.Fourth,an LPS-induced RAW264.7 macrophage inflammation model was established to study the anti-inflammatory activity of IgY and Fab fragments.The results showed that IgY and Fab fragments had no toxic effect on macrophages,and both of them could increase the activity of macrophages cell.After being stimulated with LPS,the activity of RAW264.7 cells was significantly increased,and only Fab fragments have a significant inhibitory effect on LPS-induced abnormal increase in cell activity.Furthermore,among 50-400?g/m L,Fab and IgY have significant down-regulation effects on TNF-?,IL-6 and IL-10 factors.Each factor can be down-regulated by 69.7%,77.1%and 46.1%.in addition,Fab and IgY can modulate immune activity through NO/i NOS and PGE2/COX-2 pathways,when the dosage is400?g/m L,the Fab fragment could generate the best anti-inflammatory activity.Fifth,an LPS-induced RAW264.7 macrophage inflammation model was established to study the anti-inflammatory activity of IgY and Fab fragments.The result suggested that Fab fragment among 50-400?g/m L can modulate the activities and functions of macrophages through NF-?B and MAPK signaling pathways.Fab fragment can obatin the best inhibitory effect at a dosage 400?g/m L,under which52.7%p65 can be inhibited being translocated into nucleus;while MAPKS,including p38,ERK1/2 and JNK1/2 can be inhibited to be phosphorylated at much extent of42.2%,41.5%,33.2%,44.2%and 39.6%.,respectively.These phenomenons indicated that Fab fragment inhibits NF-?B and MAPK signaling pathways in RAW264.7through interaction with the RLR4 and?V?3 integrin.In adittion,laser confocal analysis showed that the internalization of Fab fragment by endocytic mechanism may also involved in madulating the immune reaction.