Heterologous Expression,Screening,Identification And Application Of Genetic Engineering Antibody/Peptide Against 17 β-Estradiol

Environmental estrogens（EEs）are the most typical endocrine disrupting chemicals（EDCs）,which can interfere with the hormone metabolic balance in the organism and affect the physiological functions of growth,development,and reproduction.EEs pollute soil and water sources through human and animal excretion,industrial wastewater,and other ways,and inevitably circulates into the food chain.Therefore,it is particularly important to establish a rapid,accurate,and sensitive immunoassay method.One of the main tasks to solve the specificity and reliability of rapid immunological determination is to obtain immunoassay recognition elements with high affinity and specificity for specific targets.Compared with traditional antibodies,recombinant antibody technology through genetic engineering is an important means to obtain high-quality antibodies.In this paper,17β-estradiol as a model,its antibody and antibody fragment immune recognition elements were transiently expressed mammalian cell Expi293F cell.Based on the antibody,the fluorescent quantitative detection strip was prepared to realize the highly sensitive and rapid detection of 17β-estradiol in milk.In addition,through phage display peptide library combined with second-generation sequencing technology,estradiol peptide recognition elements and estradiol peptide mimic epitopes were screened.And an anti-matrix interference sensor for direct detection of 17β-estradiol in milk was constructed.The main research contents are as follows:1.Expression and evaluation of 17β-estradiol IgG antibody,Fab antibody and scFv antibody.The sequence information of 17β-estradiol antibody（PDB ID:1JN6）was obtained from Protein Data Bank（PDB）database.After codon optimization,17β-estradiol IgG antibody heavy chain and light chain expression vectors（pTT5-IgGH and pTT5-IgGL）,17β-estradiol Fab antibody heavy chain expression vector（pTT5-Fab H）,and 17β-estradiol scFv expression vector（pTT5-scFv）were constructed,respectively.Total 30μg expression plasmid was co-transfected into mammalian cell Expi293F in the ratio of heavy/light chain of 1:1.5.The transient expression of IgG,Fab and scFv antibodies were carried out respectively.The results showed that about 800μg 17β-estradiol IgG and Fab antibody were obtained through 30 mL Expi293F cell transient expression system.The expression yield is about 27 mg/L.Based on the expressed IgG and Fab antibodies,the IC₅₀ of standard curve of 17β-estradiol competitive ELISA was 0.129 ng/mL and 0.188 ng/mL,respectively.In this experiment,scFv was not obteined from the culture medium,but scFv bands were observed in the cell precipitation Lane in WB.2.Fusion expression and evaluation of 17β-estradiol Fab-EGFP antibody,scFv-EGFP antibody.In order to further explore the expression of scFv,EGFP gene was used as an indicator to connect with antibody gene,and the recombinant expression vectors pTT5-Fab H-EGFP and pTT5-scFv-EGFP were constructed.Under the same conditions,Fab-EGFP and scFv-EGFP were transiently expressed in mammalian cells.The localization and distribution of antibodies Fab EGFP and scFv EGFP in and out of cells were preliminarily detected by fluorescence intensity comparison.In order to further explore the influence of different expression vectors on antibody expression yield,pTT5 vector was replaced with pc DNA3.4and p CEP4 by seamless cloning.The signal peptide remained unchanged,and the expression vectors pc DNA3.4-IgGH/L,p CEP4-IgGH/L,pTT5-Fab H-EGFP and pc DNA3.4-Fab H-EGFP were constructed,respectively.The results showed that more Fab-EGFP and scFv-EGFP antibodies were not secreted into the culture medium.The expression yield of IgG antibody based on pc DNA3.4 and p CEP4 vector is about 0.667 mg/L and 7 mg/L.Therefore,pTT5 is more suitable for mammalian transient expression vector for IgG full-length antibody.3.Construction and mechanism research of screening methods for peptide immune elements that specifically recognize 17β-estradiol and its mimic epitope peptides.It is rare to obtain published antibody sequences from the database or literature,and most of the antibody gene information of target molecules is non-public.There are tens of millions of potentially high-risk chemical pollutants in the environment and food.In this case,how to quickly obtain the specific immune recognition elements of target molecules is one of the important tasks of rapid immune detection.In this chapter,17β-estradiol antigen（E₂-BSA）and antibody（anti-E₂mAb）were used as target molecules,and the next generation sequencing（NGS）was combined with the traditional screening method of phage display peptide library to screen the antibody peptide that specifically recognizes 17β-estradiol and the mimic epitope peptide of 17β-estradiol molecule.The whole screening process only needs one round,and there is no need for phage amplification and purification.The accuracy and success rate of screening results are greatly improved.Finally,the antibody peptides that can specifically recognize 17β-estradiol（ASNYFEHRIHLF,SAEDHQSYTAAR,HTEIGIARLLSW）and the mimic epitope peptide（KWQIWHGYFFGW,KWQIWHGYFFYW,KWQIWHGEFFGW）were obtained4.Construction of fluorescent quantitative immunochromatographic strip for detection of 17β-estradiol in milk.Immunochromatographic strip is the best carrier for developing on-site real-time detection equipment because of its small and portable size,low-cost and fast response speed.Based on quantum dot microbeads（QBs）,recombinant protein A（SPA）and rabbit anti mouse IgG antibody（anti-IgG）,the fluorescent probes（QBs@SPA and QBs@SPA@anti-IgG）were constructed.Using antibody as recognition element and fluorescent probes as signal donor,17β-estradiol immunochromatographic test strip was constructed.The detection system is improved by optimizing the overall performance of the detection probe.SPA and anti-IgG fixed on the surface of the probes can recognize the Fc of17β-estradiol antibody and fully expose its antigen recognition region.The antibody captures the analyte in a free state,and the probe captures the antibody or antibody antigen immune complex.With the decrease of 17β-estradiol concentration in the system,the fluorescent probes were enriched in the T-line and the fluorescence value increased.Under the optimized conditions,the linear relationship between estradiol concentration and T/C fluorescence specific inhibition rate corresponding to the concentration was established.The results showed that the detection ranges of E₂ based on the two fluorescent probes were 0.1–200 ng/mL and0.01–100 ng/mL,respectively,and the corresponding IC₅₀ values were 8.83 ng/mL and 0.93ng/mL,respectively.The probes are not only suitable for the detection of 17β-estradiol,but also can realize the highly sensitive detection of other targets.5.Construction of anti-matrix interference peptide sensor for detection of 17β-estradiol in milk.Matrix interference in food is an important factor affecting the accuracy of immunoassay.Antifouling materials are an effective means to solve matrix interference.The"three-in-one"multifunctional peptide chain with 17β-estradiol mimic epitope peptide,anti-matrix interference peptide and rigid structure anchor peptide was designed and synthesized,which was applied to the electrochemical sensing detection platform to construct an anti-matrix interference peptide sensor that can directly detect 17β-estradiol in milk without sample pretreatment.Under the optimized conditions,the detection range of 17β-estradiol is between0.5 pg/mL and 1 ng/mL,and the IC₅₀ is 0.0159 ng/mL.Method validation was carried out by ELISA kit.The recovery of E2 ranged from 84.3±5.9%to 104.1±3.3%.