| Type Ⅰ hypersensitivity is a common clinical disease,which is characterized by IgE-mediated local or systemic reactions caused by mast cells and basophils releasing biologically active mediators.IgE is very low in normal human serum,only 150 ng/mL,the half-life is about 3 days,but its level in the serum of patients with allergic diseases can be 10 to 1000 times higher than that of normal people and can be maintained for two weeks.For allergic diseases,there has been no satisfactory treatment so far.Desensitization therapy can be performed on patients with known allergens,but desensitization therapy takes a long time,and individual efficacy varies greatly.At present,the treatment of most moderate to severe allergic diseases mainly depends on hormone therapy,but there are side effects,which seriously affect the quality of life of patients.Studies have shown that lowering the level of IgE in the blood of patients can significantly reduce the clinical symptoms of patients with allergic diseases.Immunoadsorption(IA)therapy refers to the use of antigen-antibody binding reaction through cardiopulmonary bypass to remove the pathogenic factors in plasma or the use of adsorbent materials to remove the pathogenic factors related to immunity in plasma,so as to achieve the purpose of treating or alleviating the disease Technology,IA has the advantages of high specificity and high safety.It has been successfully applied to some classic autoantibody-mediated diseases,such as pemphigus.IA has also been used to treat allergic diseases.IA can quickly reduce the IgE level of patients.Some studies have shown that an immunoadsorption column that can bind all immunoglobulins can adsorb IgE in the blood of patients,and then use omalizumab in combination with IgE.Patients with severe allergic dermatitis with high levels have good clinical efficacy.However,the IgE immunosorbent currently used in clinical practice is mostly non-specific.Reducing the level of IgE will also reduce the level of other immunoglobulins,increasing the risk of infection of patients during treatment.This study is dedicated to the preparation of specific anti-human IgE antibodies with a view to reducing side effects during use.There is a heavy chain antibody that does not contain the light chain part and the heavy chain CHI region in the camelids.Molecularly cloning the variable region gene of the heavy chain antibody can obtain a variable domain(Variable domain only)of heavy-chain antibody(VHH)constitutes a single-domain antibody,called VHH antibody.The molecular weight of VHH antibody is only 15kD,also known as Nanobody(Nb),which is the smallest fragment known to be able to bind antigen.Its small molecular weight,stable structure,good solubility,high affinity,easy expression and purification have many advantages.Therefore,we chose to immunize alpaca to construct an anti-human IgE phage antibody library,and to screen anti-human IgE antibodies.The main contents of this study are as follows:1.Construct CHO-K1 stable transfected cell line and express a large amount of human IgE heavy chain 2-4 region(IgECc2-4)protein:before experiment,determine the optimal screening concentration of G418 for CHO-K1 cells used in this research.Dilute the cells to 1000 cells/ml,set different G418 concentrations with reference to the concentration range given by molecular clones,select the lowest G418 concentration at which all angel cells die at 10-14 to carry out the next screening experiment,and then use the laboratory pre-construction The pcDNA3.1-sp3-IgECε2-4-His plasmid was transiently transfected into CHO-K1 cells,and G418 was screened with the optimal screening concentration.After two monoclonal screenings,5 cells stably expressing the target protein were screened.Western blot was used to identify the protein expression levels of the five cells.The cell line with the highest expression level was selected for suspension and domestication.A large amount of IgECε2-4 protein was prepared by suspension expression to meet the needs of subsequent immunogens and experimental standards.2.Preparation and identification of mouse anti-human IgE monoclonal antibody:mice were immunized with the recombinant protein IgECε2-4 prepared above,and after three immunizations,mice with a titer of 1:12800 were selected for cell fusion.Cell-based cellulose semi-solid medium method for screening monoclonals to save time for cell screening.Positive clones were screened by indirect ELISA.After five cell fusions,a total of 29 positive monoclonal cells were obtained.Positive cell lines were identified by indirect ELISA and Western blot The affinity of the secreted monoclonal antibody with natural IgE and the cross-reaction with several immunoglobulins such as IgG,IgM,IgA,and IgD,Western blot and ELISA results were consistent.Two monoclonal antibodies,3E9 and 3D4,with high affinity to human IgE and basically no reaction with several other immunoglobulins were selected as candidate immunosorbents.3.Preparation and identification of alpaca anti-human IgE single domain antibody:immunize alpaca with the recombinant protein IgECε2-4 expressed above,after four immunizations,extract peripheral blood lymphocytes from the alpaca jugular vein to extract total RNA,Reverse transcribed into cDNA library.Primers were designed according to the heavy chain antibody conserved sequences,and a large number of VHH fragments were obtained by PCR amplification.At the same time,a large number of phagemid vectors pHIAT-1 were prepared.The VHH fragments and phagemid vectors were double digested with HindⅢ and Not Ⅰ.Connected and transformed into TG1 cells by electroshock to construct a Nanobody cell library.At this time,the bacterial library is the original library.The original library constructed in this study has a storage capacity of 4.3 ×109.M13K07 helper phage was added to rescue the library,and a Nanobody phage library was constructed.The library at this time can be directly used for screening.Enzyme plates were coated with human natural IgE decreasing round by round and added to the phage library for screening.After three rounds of screening,the library reached a large degree of enrichment,and 44 plates were randomly selected from the plates after the third round of screening The positive rate of the monoclonal test,44 clones were all positive clones,the positive rate was 100%,the positive clones were sequenced,and the sequencing results were analyzed with DNAMAN for the amino acid sequence,and a total of seven different sequences were found.Prokaryotic expression vector was constructed with PET-28a,and these seven antibodies were expressed with BL21(DE3)expressing bacteria.The antibodies were purified by nickel affinity chromatography.The affinity with human IgE and IgG,IgM,IgA were detected by indirect ELISA and Western blot.,IgD cross-reaction of several immunoglobulins,and finally selected SD1 and SD2 with high expression level,high affinity with human IgE,and basically no reaction with several other immunoglobulins as candidate immunosorbents.4.Selection of immunoadsorption ligands:the two mouse monoclonal antibodies and two alpaca single domain antibodies selected above were coupled to agarose by glutaraldehyde method,and the coupling efficiency and the Human IgE adsorption efficiency and cross-adsorption of IgG and other indicators,there is no significant difference in the coupling efficiency of the four antibodies,all around 90%,and the adsorption efficiency of IgE after SD2 coupling is slightly higher than that of the other three antibodies.The cross-adsorption of IgG is slightly lower than that of several other antibodies,so the single-domain antibody SD2 was finally selected as the immunosorbent for subsequent experiments.In this study,we successfully constructed a CHO-K1 cell line stably expressing IgECε2-4,and successfully screened mouse-specific anti-human IgE monoclonal antibodies and alpaca-specific anti-human IgE single-domain antibodies,binding the antibody affinity The best immunosorbent was selected for the sex,specificity,coupling efficiency and adsorption efficiency of human IgE,which provided the raw materials for the preparation of immunoadsorption columns for the treatment of allergic diseases. |
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