Preparation And Activity Assay Of The Recombinant Protein,Single Chain Antibody Against Human Insulin And Enhanced Green Fluorescent Protein(scFv-EGFP)

Recombinant monoclonal antibodies,which specifically target clinical biomarkers,have increasingly been used as powerful tools in diagnosis for many diseases.Green fluorescent protein?GFP?as fluobody and its red-shifted mutant,enhanced green fluorescent protein?EGFP?,which fluoresces 35 times more intensely than the wild-type GFP,have been used as fluorescent markers to observe protein localization in vivo and in vitro studies.Genetic fusion of GFP/EGFP to antibodies has led to the expansion of fluorescent antibodies,which have been used as a credible detection tool for in vitro studies and for tumor imaging.Proinsulin was used as a target antigen and is being widely used in the management of diabetes.Recently,several scFvs against proinsulin were used for the construction of immunoassays,which specifically target insulin substances in serum and blood.In this thesis,the recombinant proinsulin and scFv-EGFP fusion protein were prepared and tested for their activity.The main research results were as following:?1?The E.coli BL21 pET-Ins strain was cultured at 30°C,220 rpm to an OD₆₀₀?0.6,and then induced with 1mmol/L IPTG for 4h at 26°C,220rpm.Analysis by SDS-PAGE showed that there was no significant accumulation of recombinant human proinsulin in the centrifuged supernatant after disruption.In the centrifuged pellet after disruption,recombinant human proinsulin protein accounted for 49.9%of the total precipitated protein.This reveal that human proinsulin is mainly present in the form of inclusion bodies.?2?Human proinsulin protein was extracted and dissolved with urea and inclusion body washing solution respectively.SDS-PAGE analysis showed that the purity of human proinsulin from the inclusion bodies was 84.4%.The purity of human proinsulin from the washed and re-solved inclusion bodies was 99.8%.The yield of the fusion protein is about197.12mg/L.?3?The nuclear DNA of transgenic P.pastoris GS115 scFv-EGFP from 1 to 6 strains was extracted and amplified by a pair of primers,SEP1 and SEP2,between 1208 and 1724 bp?total length 516 bp?of the scFv-EGFP coding fragment.The results showed that P.pastoris GS115 scFv-EGFP 1-6 strains present a clear,specific band with a size of 500 bp.?4?The cells of the transgenic P.pastoris scFv-EGFP-6 and P.pastoris GS115 strains were analyzed via fluorescence microscopy.The fluorescence of the recombinant protein was visible in the entire cytoplasm of P.pastoris scFv-EGFP-6 strain.?5?The P.pastoris GS115 scFv-EGFP strains 1,2,3,4,5,6,7,8,9,10,11 and 12 were cultured at 30?and 220 rpm for 96 hours.The proteins from the precipitated fermentation broth and from the disrupted cell pellets were analyzed by SDS-PAGE electrophoresis.The electrophoresis results showed that strains 1 to 12 had specific bands near 54.6 kDa?theoretical molecular weight of scFv-EGFP?.The band proteins accounted for 30.3%,11.1%,19.1%,60.6%,63.2%,68.1%,35.4%,3.8%,4.1%,10.8%,7.1%and 15.9%of the total proteins in the cell pellets after disrupted by ultarsonication?in the order of 1 to 12 strains?.The proteins from the fermentation broth were firstly precipitated by acetone and then analyzed by SDS-PAGE.The protein bands near to 54.6 kDa accounted for 30.0%,14.8%,23.3%,31.7%,30.4%,27.3%,35.3%,18.4%,14.1%,0.0%,5.9%and 0.0%of the total proteins in fermentation broth?in the order of 1 to 12 strains?.?6?The scFv-EGFP fusion protein-containing samples were detected by the western blotting,and find that the scFv-EGFP sample can be bound by the antibody against GFP.?7?The supernatant of P.pastoris GS115 scFv-EGFP-6 cells was precipitated by 30%,40%and 50%of the ammonium sulfate.After centrifugation,the prepared solutions after fractional precipitation was further purified on a Ni-NTA affinity column.The results showed that the protein bands near 54.6kDa did not appear in the elution buffer.These results suggested that the recombinant fusion protein scFv-EGFP may not bind to Ni-NTA.?8?According to result?7?,we re-checked the pGAP?A-scFv-EGFP vector sequence and found that there is a frameshift mutation at the end of the scFv-EGFP coding fragment,and a stop codon TAG appeared before the Myc and His tags.The recombinant fusion protein scFv-EGFP did not contain a 6xHis tag.The purification results for the other strains were similar to those of the No.6 strain,and it was suggesting that the stop codon had appeared before the construction of the transgenic yeast.?9?According to results?7?and?8?,we used ion exchange resin DEAE?DE-52?to purify the recombinant fusion protein scFv-EGFP.After disrupted the cells by ultrasonication,the supernatant of P.pastoris GS115 scFv-EGFP 6 strain was equilibrated with 40mM Tris-HCl?pH 6.5?and added to the column.Proteins were eluted by increasing of 1-2M NaCl buffer?the pH 5.31 as the protein isoelectric point?pI??.and then we measured the optimal density?OD?of the elution fractions the absorbance was at 280 nm.The maximum absorbtion peak appeared in tube 1.SDS-PAGE analysis of the fractions samples from tube 2-9 showed specific bands near 54.6kDa?theoretical molecular weight of scFv-EGFP?.The band proteins accounted for 62.3%,50.1%,28.8%,28.8%,17.7%,15.4%,7.4%,17.9%,respectively.In addition,there are other proteins bands were present in addition to the 54.6 kDa protein band.?10?To detect the sensitivity of scFv-EGFP fusion protein to proinsulin,we used human proinsulin with different concentration?4.1ng/ml 4.5ng/ml,5.9ng/ml and 8.6ng/ml?detected by scFv-EGFP?7.9ng/ml?protein and showed a good dots fluorescence.Negative control?E.coli BL21?DE3??did not show exposure signal or fluorescence,this indicates that the recombinant fusion protein scFv-EGFP has specific binding to human proinsulin.