The objective of this study is to generate broad spectrum monoclonal antibody ( mAb ) against a group of pyrethroid insecticides and to identify its immunological characteristics. The generic hapten 3-phenoxy-benzoic acid ( PBA) was conjugated to carrier protein BSA by activated ester method. Balb/c mice were immunized with PBA-BSA. The titer of polyclonal antibody ( pAb ) was detected by indirect enzyme-linked immunosorbent assay ( ELISA) after five times immunization. The mouse with high titer and sensitivity was selected for cell fusing. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by indirect non-competitive ELISA based on the coating antigen PBA-ovoalbumin ( PBA-OVA ) . High-sensitivity and high-specificity mAb was prepared after subcloning using limiting dilution method. Purified mAb was obtained after purified by saturated ammonia sulfate precipitation and protein G affinity column. The immunological characteristics of mAb such as titer, antibody subtypes, affinity constant and the sensitivity to pyrethroid insecticides were characterized by indirect ELISA; The results of UV spectroscopy and SDS-PAGE showed that PBA-BSA artificial antigen was synthesized successfully. A hybridoma cell line (4H11) secreting anti-pyrethroid mAb was established. The titre of ascites was up to 1:6. 5×106, and the mAb was IgG1 subtype. The affinity constant of the mAb to PBA was about 2. 5×107 L/mol, with a IC50 value of 208. 83 μg/L and a detection limit of 21. 23 μg/L to PBA. Simultaneously, beta-cypermethrin, flucythrinate, cypermethrin and fenvalerate were sensitively recognized by the mAb with the IC50 of 1. 01, 2. 15, 3. 16 and 3. 67μg/L, respectively.

Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3'-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 3'-PO4 end of the substrate and generates 3'-OH,TdT can effectively elongate the 3'-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 3'-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10-3 U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.

Objective:To understand the results of blind evaluation of dissertation of three-year doctors and eight-year medical doctors, and to explore the improvement measures of eight-year program education.Methods:The data analysis method was manipulated. A total of 47 eight-year doctoral and 88 three-year doctoral dissertations submitted by the first clinical college of Huazhong University of Science and Technology in 2020 were selected as the research material. SPSS 17.0 was used to perform Chi-square test to compare the itemized evaluation opinions of the dissertation, Spearman test was used to analyze the correlation between the defense opinions, itemized evaluation opinions and the overall evaluation.Results:The gap between eight-year and three-year doctoral dissertation is mainly manifested in innovation and research value ( χ2=9.10, P=0.003), topic and review ( χ2=5.70, P=0.017), while there is no significant difference in the overall assessment and oral defense suggestion. The main influencing factor of dissertation defense suggestion for both doctors was the dissertation standardization (eight-year: r s=0.53, P<0.001; three-year: r s=0.45, P<0.001). The evaluation results of eight-year doctor dissertation were closely related to basic knowledge and scientific research ability ( r s=0.74, P<0.001). Conclusion:There is no significant difference between eight-year doctors and full-time doctors in research attitude. But there was a certain gap in scientific research and innovation ability among them. It is suggested to clarify the teaching objectives, formulate and refine the evaluation system of dissertations, and strengthen the cultivation of scientific research interest and academic belief of eight-year study program.

Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2β anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2β anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.
Insecticides/metabolism* ; Bacillus thuringiensis ; Endotoxins/pharmacology* ; Bacillus thuringiensis Toxins/metabolism* ; Hemolysin Proteins/pharmacology* ; Bacterial Proteins/chemistry* ; Plants, Genetically Modified/genetics* ; Pest Control, Biological