Development And Preliminary Application Of Anti-recombinant Human Interferon α2b Nanobody

Research background Hepatitis is a prevalence endemic in China,recombinant human interferonα2b is a world recognized treatment for hepatitis.As recombinant therapeutic proteins,more attention should be paied to the quality control of them.The identify experiments are the main evidence for the qualitative detection.The recombinant human interferon α2b is identified by an immunological method（immunodoting or Western Blot）according the Chinese Pharmacopoeia.This immunological method takes long time and cost a lot.Antibody used in this method different from one batch to batch,and they don’t satisfy the demand of anti-counterfeit of recombinant human interferon α2b for their requirement of cold chain transportation.In this subject an anti-recombinant human α2b nanobody named I₂₂ was successfully expressed through prokaryotic express system.This nanobody is more sensitive,economical and can resist extreme temperature.We have used this nanobody to develop a colloid gold method to realize rapid identify of recombinant human interferon α2b.Method 1.The alpaca was repeatly immunized with recombinant human interferon α2b protein and monitor the serum antibody titer.It showed that after third time of booster immunization,the serum antibody titer was obviously increased.We collected 25 m L blood of Alpaca after the fivth immunizations.We separated the lymphocytes from Alpaca’s blood,and extracted total RNA from the lymphocytes that we obtained.After the lymphocytes were separated,the total RNA was extracted,and the universal primers were used to amplify the VHH gene region throuth a nested PCR technology,by a strains of enzyme digestion,connection and electrical transformation.2.A phage library was constructed and repeatly screened for anti-recombinant human interferonα2b nanobody in vitro.A anti-recombinant human interferonα2b nanobody was obtained though phage display technique,The nanobody was named I₂₂,some corresponding validation were conducted to confirm the ability of I₂₂.3.PET28a-His-I₂₂-His,p ET28a-His-I₂₂ and p ET28a-I₂₂-His three plasmids were successfully constructed to express the nanobody in prokaryotic.4.Some physicocal and chemical properties of the nanobody I₂₂ were studied,including the isoelectric point,N terminal sequencing,the mass spectrum,and we also evaluated whether nanobody I₂₂,commercial monoclonal antibody and commercial ployclonal antibody can affect biological activity of recombinant human interferon α2b when bind to it,or there was any side effect to the recombinant human interferon α2b biological activity.These studies laid foundation for the research on the application of nanobody I₂₂.5.After the nanobody I₂₂ were placed at room temperature,the nanobody I₂₂ was used in an immunoblotting experiment,and the sensitivity of I₂₂ was compared with a commercialized monoclonal antibody.We have used I₂₂ to establish a Semi quantitative detection method,and I₂₂ was applied to develop a gold labelling immunological assay.Result 1.The recombinant human interferon α2b nanobody gene library with a capacity of 2.6×108 was obtained.The phage display library of anti-recombinant human interferon α2b nanobody was obtained after the expression of phage display technique,and the titer of the recombinant human interferon α2b nanobody library was 3.1×1013CFU/ m L.2.The positive clones were verified by ELISA and Western Blot experiments,and two positive clones were obtained by ELISA verification.The positive clones were sequenced,and these two positive clones share the same genome sequence.We name the nanobody expressed by the positive clone as I₂₂.Western Blot experiment verified that the supernatant of positive clones could specifically bind to recombinant human interferon α2b.3.We constructed three expression plasmid named PET28a-His-I₂₂-His,p ET28a-His-I₂₂ and p ET28a-I₂₂-His and obainted some proteins featured nearly the same molecular weight by expressing these three strains,these proteins can be binding with recombinant interferon α2b with no signifinant distinction.We choose protein expressed by p ET28a-His-I₂₂-His strain in the subsequent experiments,the yield of the protein was 45.7mg/L and the purity of the protein was 100%.4.The isoelectric point of the nanobody was 8.20.The amino acids detected by N terminal sequencing was in accordance with the theoretical results,and the molecular weight measured by mass spectrometry was 14226.80 Da,which also correspond with the theoretical molecular weight of 14226.77 Da.It has been proved that the nanobody I₂₂ and the commercialized monoclonal antibody had no effect on the biological activity of recombinant human interferon α2b drugs when binding to them,meanwhile the polyclonal antibody had an effect on the biological activity of recombinant human interferon α2b drugs when binding to them.5.The nanobody I₂₂ was used in a Western Blot experiment,and was specific to the recombinant human interferon α2b drugs.There was no binding ability to the excipients such as Human Albumin in recombinant human interferon α2b drugs.Conclusion The gene sequence of anti-recombinant human interferon alpha 2b nanobody was obtained unprecedently,and the protein was soluble expressed in a prokaryotic system of E.coli.The nanobody I₂₂ was used in an immunoblotting experiment,and the sensitivity of I₂₂ was compared with a commercial monoclonal antibody.Results showed that nanobody I₂₂ was more sensitive,stable and economical.The production of commercialized antibody was about 25mg/L.The production of nanobody is about 45.7mg/L,purity of purification by nickel column is 100%.In order to realize rapid detection of recombinant human interferon α2b,satisfy the demand of anti-counterfeit of recombinant human interferon α2b,we used I₂₂ to establish a colloidal gold labeling immunological assay.The LOD of colloidal gold labeling immunological assay can reach up to 1μg/m L to realize preliminary application of antibody in rapid detection.