| In alpacas,there is a heavy chain antibody,and removing the variable region of such antibodies is a nanobody,are the smallest naturally occurring antigen binding domains.Because of their high stability,refoldability,and manipulable characteristics,VHHs have been developed for therapeutic,diagnostic,bioimaging,and immunoassay.Efforts have been made to incorporate VHHs into biomaterials as recognition elements for affinity purification.The capacity of the adsorption resins or materials is an important feature for affinity chromatography.Higher adsorption capacity is a benefit to lower the cost and reduce the time for the downstream process of purification,especially in industrial applications.To increase the adsorption capacity,the key is to improve the bioactive molecular density of the recognition ligand.The paper improves the bioactive molecular density of the recognition ligand by tandem anti-mouse Fc nanobodies,it is important for the research.The main experimental results of this study were as follows:1.Through gene engineering technology and principle,anti-Fc nanobody AFV were fused the H-AFV by using Rigid Linker(MAQPA),and tandem them by sfi I restriction endonuclease.Instruct expression vector pET30a-H-AFV,pET30a-H-diAFV,pET30aH-triAF and pET30a-H-tetAFV fused Halolinker,after colony PCR verification,it was sent to the gene sequencing company for sequencing.Comparison with the original sequence,AFV was inserted into the predetermined position correctly,and the gene did not mutate.2.The previous description vectors transformed into the BL21(DE3)competent cell,inducing protein expression at low temperature with IPTG,using protokaryon expression system expressed protein.SDS-PAGE analysis pre-induce and post-induce bacterial solution and fusion protein by Ni-NTA purification.Results indicate that it was successfully expression soluble protein:H-AFV?H-diAFV?H-triAFV and HtetAFV.Confirm that four kinds of protein the best induce condition was 18,220rpm inducing 10 h by optimizing expression condition.3.Test interaction bioactive mouse IgG with indicate protein,indirect ELISA was performance that find H-tetAFV has the highest affinity than other recombination protein,and KD value is the highest 42 nM by Biolayer interferometry(BLI)test,the other proteins especially were 77.3nM 59.2nM and 50.4nM proteins molecular range from high to low.Kinetic analysis confirms the enhancement of avidity is mainly contributed by the ascending of the association constant(Kon).4.The preparation process of oriented immobilization immunoaffinity column was optimized,and the static adsorption capacity of the immunoaffinity column changed with the constant change of the nanobody input.Four the affinity column prepared by H-diAFV and H-tetAFV proteins,the static adsorption capacity reached the highest value when the input was 6 mg mL-1.Once the amount of input exceeds this value,the static capacity decreases.When the amount of H-AFV protein and H-triAFV protein was more than 8 mg mL-1 and 5 mg mL-1 respectively,the static adsorption capacity decreased.5.The maximum static capacities of H-AFV,H-diAFV,H-tri AFV and H-tetAFV were 7.53±0.41 mg mL-1,13.8±0.59 mg mL-1,7.5±0.86 mg mL-1 and 12.76±0.66 mg mL-1,respectively.It can be observed from the above data that the static capacity of H-diAFV and H-tetAFV increased by 83.3%and 69.4%,respectively.However,with the increased of valence,the static capacity of H-tri AFV did not increase.It was initially suspected that the spatial structure affected the binding of H-triAFV to mouse IgG.According to the performance of affinity column prepared by H-diAFV and H-tetAFV,the elution rate of mouse IgG could still reach over 75%after repeated use for 10 times.Different concentrations of NaOH(1N,0.5N,0.1N)were used to wash the affinity column to test its static adsorption capacity.It was found that affinity column prepared by H-triAFV could withstand higher alkalinity,and the final adsorption capacity could still reach 32.6%of the original under the condition of 0.1N NaOH. |
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