Preparation And Activity Analysis Of The Human Insulin Single Chain Antibody And HRP Recombinant Protein

As people’s lifestyle changes,the incidence of diabetes continues to rise.Changes in serum insulin in diabetic patients are mandatory items for the diagnosis and classification of diabetes types.A quick,convenient and reliable method of examination is undoubtedly a necessary means of confirming the cause of diabetes.The traditional enzyme-linked immunoassay method involves two molecular hybridizations,which takes a long time in practical application such as clinical testing,and increases the labor intensity of the operator.The recombinant fusion protein sc Fv-HRP of primary antibody(monoclonal antibody)and the labeled enzyme horseradish peroxidase was constructed by genetic engineering,and the two-molecular hybridization(primary antibody binding to insulin and secondary antibody combined with primary antibody)was simplified into primary molecular hybridization.(Insulin combined with recombinant fusion protein)can greatly shorten the length of enzyme-linked immunoassay,reduce the labor intensity of workers,and improve detection efficiency.The main findings of this paper are as follows:(1)Using the transgenic yeast P.pastoris GS115 sc Fv-HRP 1 preserved in our laboratory,the nuclear DNA was extracted,and the sc Fv-HRP coding fragment was amplified by primer SHP1 and primer SHP2 from 1210 to 1780 bp(total length 570 bp).Area to determine if the strain is positive.The results showed that P.pastoris GS115 sc Fv-HRP 1 amplified a clear,specific,slightly larger than 500 bp band.(2)Incubate P.pastoris GS115 sc Fv-HRP strains 1,2,3,4,5,6,7,8,9,10,11 and 12 for48 h at 30 ° C and 220 rpm to extract the fermentation broth and cells.The precipitated protein was analyzed by SDS-PAGE electrophoresis.The electrophoresis results showed that strains 1 to 12 all had specific bands near 63 k Da(the theoretical molecular weight of sc Fv-HRP).The strip protein accounted for 15.6%,16.6%,15.1%,1.9%,15.1%,11.5%,1.2%,0.8%,14.1%,16.0%,16.6%,and 8.4% of total protein in the cell,respectively.Strain Nos.1to 12);the protein content of the strip protein in the fermentation broth was 55.0%,51.9%,47.3%,0%,5.7%,82.2%,0%,0%,23.7%,62.2%,respectively.59.4% and 11.1%(in order of strains 1 to 12).(3)P.pastoris GS115 sc Fv-HRP 1 cells were collected by sonication,and the supernatant after centrifugation was precipitated with 50% saturated ammonium sulfate.The preparative solution after fractional precipitation was further purified on a Ni-NTA affinity column.The results showed that the protein band near 63 k Da was up to 33.9% in the wash buffer and was essentially absent in the elution buffer.This result suggests that the recombinant fusion protein sc Fv-HRP may not bind to Ni-HTA.(4)In view of the results of(3),we re-checked the p GAP-sc Fv-HRP vector sequence and found that there is a frameshift mutation at the end of the sc Fv-HRP coding fragment,and a stop codon TAG appeared before the Myc and His tags.Thus,the recombinant fusion protein sc Fv-HRP did not contain a 6x His tag.The purification results for the other strains were similar to those of the No.1 strain,and it was speculated that the stop codon had appeared before the construction of the transgenic yeast.(5)In view of the results of(3)and(4),we used an anion exchange resin DEAE(DE-52)to purify the recombinant fusion protein sc Fv-HRP P.pastoris GS115 sc Fv-HRP strain 1 was centrifuged on the supernatant,and the column was washed with p I5.3 elution buffer.The eluent was collected and the absorbance at 280 nm was measured.The maximum absorption peak appeared.In the 6th tube.SDS-PAGE analysis of samples collected from tubes 3-9showed that other protein bands were present in addition to the 63 k Da protein band.(6)Horseradish peroxidase activity in the supernatant of P.pastoris GS115 sc Fv-HRP strain 1-12 was determined using ABTS and guaiacol as substrate.The results of the ABTS assay showed that the enzyme activities in samples 1-12 were 1.77 U/m L,3.25 U/m L,4.15U/m L,14.57 U/m L,13.15 U/m L,and 10.80 U/m L,respectively.10.26 U/m L,6.79 U/m L,11.13 U/m L,6.98 U/m L,10.06 U/m L,8.94 U/m L,and the control was 0.02 U/m L.The results of the determination of guaiacol as a substrate showed that the enzyme activities of samples No.1-12 were 5.41 U/m L,10.98 U/m L,6.59 U/m L,9.09 U/m L,7.13 U/m L,and11.92 U/m L,5.10 U/m L,6.82 U/m L,7.37 U/m L,2.98 U/m L,5.96 U/m L,3.84 U/m L,,respectively,and the control was 0.01 U/m L.(7)E.coli BL21 p ET-Ins strain was cultured at 30 ° C,220 rpm to OD600 = 0.6,and then induced with 1 mmol / L IPTG for 4 h at 26 ° C,220 rpm.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 55.3% of the total precipitated protein.That is,human proinsulin is mainly present in the form of inclusion bodies.(8)Extraction and dissolution of human proinsulin inclusion bodies using urea and inclusion body washing solutions,respectively.The SDS-PAGE analysis showed that the purity of human proinsulin protein extracted and dissolved by urea was 86.5%,and the purity of human proinsulin protein was 71.3% after extraction and dissolution of inclusion bodies with inclusion body washing solution.(9)Dot blotting was carried out using the above human proinsulin protein dot nitrocellulose membrane(NC membrane),recombinant fusion protein sc Fv-HRP expressed by P.pastoris GS115 sc Fv-HRP 11 strain,and positive spots appeared(negative control did not appear Exposure spots).This indicates that the recombinant fusion protein sc Fv-HRP has specific binding to human proinsulin and horseradish peroxidase activity.