Construction Of Uniconazole Complete Antigen Mimics Based On Antigen Mimotope And Its Preliminary Application In Immunoassay

Uniconazole,as a plant growth regulator,is widely used in wheat,peanuts,and other crops.Toxicological studies on uniconazole have shown that long-term use of uniconazole will have side effects on the endocrine of humans and animals,and there is a possibility of teratogenicity and mutagenicity,which poses a potential risk to human health.In the process of constructing the uniconazole immunoassay method,the preparation of complete antigen is mainly by chemical synthesis method,which faces the problems of low efficiency,high cost,many by-products,and large batch error.In this study,the preparation of uniconazole antigen mimotopes based on phage display peptides was carried out by using phage display peptide library affinity panning technology with uniconazole monoclonal antibody as the target.On this basis,the antigen mimotope（polypeptide）was fused with maltose binding protein（MBP）and thioredoxin（Trx A）based on prokaryotic expression based on genetic engineering technology.The polypeptide-fusion protein was used to replace the traditional chemical synthesis of uniconazole to detect the whole antigen.At the same time,the preliminary application of polypeptide-fusion protein（detection of whole antigen mimics）in immunochromatographic test strips was carried out,and its feasibility in a homogeneous immunoassay system was preliminarily verified.The main research results are as follows:1)Using the monoclonal antibody against uniconazole as the target,three rounds of affinity panning were carried out in the Ph.D.（?）-12 Phage Display Peptide Library.Through indirect Phage-ELISA,indirect competitive Phage-ELISA,and DNA sequencing,two antigen mimotopes of uniconazole were obtained from the third affinity panning process,named 3-25 and 3-19,respectively.The standard curve of competitive inhibition of Phage-ELISA was established.The half inhibitory concentration(IC₅₀)was 0.54 ng/m L and 0.28 ng/m L,respectively.Compared with the ELISA based on the uniconazole detection antigen prepared by chemical synthesis(IC₅₀=3.88 ng/m L),the sensitivity was increased by nearly ten times.2)The antigen mimotope（peptide）-fusion protein expression vectors were successfully constructed,which can be fused with maltose binding protein（MBP）and thioredoxin（Trx A）,respectively.They were named p MAl-polypeptide（3-19）-MBP,p MAl-polypeptide（3-25）-MBP,and p ET-polypeptide（3-25）-Trx A@Spy Tag,respectively.They were transformed into E.coli.Through colony concentration optimization and IPTG concentration optimization,the soluble expression of the polypeptide（3-19）-MBP,polypeptide（3-25）-MBP,and polypeptide（3-25）-Trx A@Spy Tag fusion protein was realized.The expression level of（3-25）-Trx A@Spy Tag was 17.28 mg/L,and the expression level was nine mg/L,the peptide（3-19）-MBP,peptide（3-25）-MBP,and peptide（3-25）-Trx A@Spy Tag prepared by biosynthesis were used as uniconazole whole antigen mimics to establish competitive ELISA standard curves.The IC₅₀ values were 14.86 ng/m L,3.20 ng/m L,and 17.34 ng/m L,respectively.the results of accelerated storage experiments at 37°C showed that under the treatment conditions of 120 h,the immunoassay performance of the three biosynthesized uniconazole whole antigen mimics did not decrease significantly,indicating that the biosynthesized uniconazole whole antigen mimics can be used as a substitute for traditional chemical synthetic-detection antigens for the construction of immunoassay methods.3)The signal probes were prepared by labeling uniconazole antibodies with colloidal gold,latex microspheres,and time-resolved fluorescent microspheres,respectively.The uniconazole antigen mimics（3-25-Trx A@Spy Tag,3-25-MBP）were fixed on the T line of the nitrocellulose membrane,and the uniconazole immunochromatographic test strips based on three signal modes were constructed.The results showed that the immunochromatographic test strips based on colloidal gold probes could not achieve effective detection.It was speculated that the signal fragment of colloidal gold could not effectively respond to the detection signal;when3-25-Trx A@Spy Tag was used as the detection antigen,the signal sensitivity of the immunochromatographic test strip based on latex microspheres was stronger than that based on 3-25-MBP,and the cutoff value was 100 ng/m L.When the time-resolved fluorescent microspheres were used as the detection signal,the IC₅₀ of the uniconazole immunochromatographic test strip with 3-25-Trx A@Spy Tag as the T-line detection antigen was 8.83 ng/m L,and it had good specificity.4)Based on the Spy Tag/Catcher self-assembly system,the reaction temperature,reaction time,and Catcher:Spy Tag molar ratio were optimized by orthogonal experiments.Based on the optimized self-assembly conditions,the fluorescent microspheres coupled with uniconazole antigen mimics（3-25-Trx A@Spy Tag）were prepared.Combined with the uniconazole antibody immunomagnetic beads,the homogeneous immunoassay competitive standard curve of uniconazole was preliminarily constructed.The SC₅₀ value was 34.82 ng/m L,and the linear range was20.05～432.7 ng/m L.The experimental results preliminarily verified the feasibility of constructing the homogeneous immunoassay method based on antigen mimics,but further research is needed.