Based On ZZ Affnity Peptide As The Key Component To Construct Recombinant Proteins Used In Immune Analysis And Research Of Antibody Purification

Objectives:Using the IgG-binding characteristics of ZZ affinity peptide,to construct and express the fusion protein between ZZ affinity peptide-alkaline phosphatase（ZZ-AP）and ZZ affinity peptide-Cys（ZZ-Cys）,exploring the feasibility of as the genetic engineering of immunoreagent used in enzyme immunoassay and ZZ-Cys applied in antibody purification.Methods:（1）Constructing pEZZ-AP vector by molecular biological techniques,the vector was transformed and expressed in E.coli DH5α,using Metal Ion Affinity Chromatography（IMAC）to purify the target protein,and alyzsing it by SDS-Polyacrylamide Gel Electrophoresis（SDS-PAGE）,the biological activities was detected by Western blot and applied to immunocytochemistry（ICC）as an alterative second antibody.（1）Different final concentrations of glycine,Triton X-100 and sucrose were added and cultivated at 28°C for 48 h.Quantitative analysis the yield of fusion protein ZZ-AP by Competitive enzyme-linked immunosorbent assay（ELISA）,the determination ofβ-galactosidase and OD600,and electron microscope were used to research the effect and mechanism of the chemicals.（3）To construct and express ZZ-Cys fusion protein,Fourier Transform Infrared Spectroscopy（FT-IR）analysis Sepharose 6-FF which was modified with epichlorohydrin,ethanediamine and maleicimidecaproicacid,ZZ-Cysfusionproteinconjugatedwith maleimide-Sepharose in oriented manner,namely Sepharose-ZZ^SA.For comparison,ZZ-Cys fusion protein was conjugated with epoxy-Sepharose in randomly manner,namely Sepharose-ZZ^RA.The amount of protein ZZ-Cys linked to Sepharose,the capacities of Sepharose-ZZ^SAA and Sepharose-ZZ^RAA were determined and compared using bicinchoninic acid（BCA）protein assay,applications of Sepharose-ZZ^SAA for IgG purification.Results:（1）The result of SDS-PAGE showed that the fusion protein with a relative molecular weight was consistent with the theoretical values（62 kDa）.The fusion protein has rabbit IgG-binding ability and enzymatic activity of alkaline phosphatase,those were validated in Western blot.（2）SDS-PAGE showed that ZZ-AP were gathered in periplasmic space,β-galactosidase activity assay,OD600values and SPSS 17.0 analyze the amount of protein expression,they were indicated that glycine,Triton X-100 and sucrose could have impact on the bacteria growth.Maintaining cells integrity,adding any two or three all can effect the the yield of extracellularly.When the culture medium contained 5%sucrose,1%glycine,and 1%Triton X-100,the maximum secretion of ZZ-AP protein reached 18.6 mg/l,which was approximately 18.6-fold more than that of the control group.TEM indicated that sucrose could increase the osmotic pressure and led to additional leakage of soluble recombinant proteins from the periplasmic space,glycine was incorporated into peptidoglycan and disrupted the peptidoglycan layer,and Triton X-100 exerted its major effect on the integrity of the outer membrane by disrupting the lipid bilayer.（3）The analyse of FT-IR showed the rection of Sepharose 6-FF was successed,IMAC and HPLC indicated that ZZ-Cys protein could bind with two IgGs,BCA showed that approximately 1.19 mg ZZ-Cys is present per gram of wet Sepharose in Sepharose-ZZ^SAA and 0.97 mg/g of wet Sepharose in Sepharose-ZZ^RA,the maximum saturation binding capacities were approximately 23.80 mg IgG/g of wet Sepharose-ZZ^SAA and 12.31 mg IgG for Sepharose-ZZ^RA,indicate that compared with Sepharose-ZZ^RA,binding capacities of Sepharose-ZZ^SAA have been improved while they have similar numbers of ligands.The result of antibody purification of Sepharose-ZZ^SAA indicated that it can apply in antibody purification.Coclusions:（1）The ZZ-AP fusion protein was constructed successfully,it has rabbit IgG-binding ability and enzymatic activity of alkaline phosphatase.We anticipate that the ZZ-AP fusion protein has a potential application in immunoassay field.（2）Compared with normal condition,the culture medium contained 5%sucrose,1%glycine,and 1%Triton X-100,the maximum secretion of ZZ-AP protein reached18.6 mg/l,which was approximately 18.6-fold more than that of the control group.（3）The modification of Sepharose 6-FF were successed,ZZ-Cys fusion protein was loaded on maleimide-Sepharose in oriented manner,compared with epoxy-Sepharose which was in randomly manner（12.31 mg Ig G/g of wet Sepharose）,the binding capacities can reach about 23.80 mg IgG/g of wet Sepharose,and can apply it in antibody purification.