Objective To develop a glucagon-like-peptide-1 receptor (GLP-1R)/gastric inhibitory polypeptide receptor (GIPR)/glucagon receptor (GCGR) triagonist peptide with balanced activity and strong resistance to dipeptidyl peptidase Ⅳ(DPP-Ⅳ) degradation using deep learning and structural modeling.Methods Based on clinical candidate Peptide 20 (MAR423), virtual mutagenesis was performed on the first 3 residues at the N-terminus， trireceptor complex stability was scored using Protein Message-passing Neural Network, and 800 sequences were screened based on the score and DDP-Ⅳ sensitivity predicted by neural network model. Top candidates were evaluated by 5 ns molecular dynamics simulations to calculate binding energy, and the median effective concentration (EC₅₀) was measured with HEK-293 reporter gene assay. DPP-Ⅳ resistance was assessed by measuring residual activity after enzyme treatment.Results Peptide A, modified with γGlu-C16, had stable conformation, with binding energy values to GLP-1R, GIPR, GCGR at (﹣151.49 ± 15.87), (﹣122.79 ± 23.42), (﹣139.25 ± 19.81) kcal·mol^﹣¹，respectively, and showed strong and balanced activity with EC₅₀ of 0.069, 0.089, 2.730 nmol/L, respectively. It retained (104.30±3.23)% activity after 24 h of DPP-Ⅳ treatment.Conclusion A rapid and effective strategy to discover triagonist peptide with improved stability and balanced potency is presented, supporting future development of multi-target peptide drugs for metabolic diseases.

Objective To observe the toxic reaction of aluminum hydroxide adjuvant intramuscular injection in mice.Methods In 50 specific pathogen free grade KM mice, preliminary experiments were performed with 10 female mice, and the remaining 40 mice (half males and half females) were randomly divided into treatment group and saline control group. High concentration (13 mg/mL) aluminum hydroxide adjuvant was administered by single intramuscular injection at dosage of 260 mg/kg. Mortality and toxic reactions were observed within 14 d after administration, and gross anatomy of systemic organs and histopathological examination of lesions were performed at the end of the study.Results The 20 mice in treatment group moved normally after intramuscular injection in the leg, and there was no death within 14 d.The body weight of female mice in treatment group was statistically significantly lower than that of control group on the 2nd and 3rd day after administration (t values were 2.23 and 2.61, respectively, P < 0.05), and the body weight of male mice in treatment group was statistically significantly lower than that of control group on the 2nd,3rd,7th and 14th day after administration (t values were 4.04, 3.93, 2.25 and 3.04, respectively, P < 0.05). The food intake of mice in treatment group was statistically significantly lower than that of control group on the 1st day after administration (t = 4.21, P < 0.01), and the water intake of mice in treatment group was statistically significantly lower than that of control group on the 1st and 3rd day after administration (t values were 3.29 and 3.49, respectively, P < 0.01). A white nodule with ~2 mm diameter was observed at the administration site of mice in treatment group, and no obvious pathological changes were observed in other tissues. Pathological examination showed that mice in treatment group presented partial degeneration and necrosis of muscle fibers, infiltration of inflammatory cells,partial proliferation of connective tissues and formation of a small number of megakaryocytes， as well as a small amount of bleeding at the injection site. The mice in control group showed no abnormality during the experiment.Conclusion A single intramuscular injection of high-dose aluminum hydroxide adjuvant in mice can cause muscle irritation at the injection site to a certain extent, food intake and water consumption reduction, and body weight loss, but no other abnormalities.

Objective To investigate the physicochemical properties of aluminum phosphate adjuvants subjected to different numbers of autoclaving cycles and to evaluate the effect of aluminum phosphate adjuvant with varying autoclaving cycles on immunogenicity of type 6A pneumococcal polysaccharide-tetanus toxoid (TT) conjugate (6A-TT).Methods Self-prepared aluminum phosphate adjuvants were autoclaved 1, 2, and 3 times, respectively. The particle size and distribution, pH, isoelectric point, free phosphate content, phosphorus-to-aluminum molar ratio, and protein adsorption rate were examined. Additionally, the effect of aluminum phosphate adjuvants treated with different autoclaving cycles, when combined with 6A-TT, on the specific antibody titers in mice was assessed.Results After autoclaving 1, 2, and 3 times, the 50% particle distribution diameters (Dv50s) of aluminum phosphate adjuvant were 5.04, 5.22, and 5.12 μm, while Dv90s were 9.20, 9.87, and 10.60 μm, respectively. The pH values were 4.6, 4.3, and 4.3, while the isoelectric points were 5.48, 5.36, and 5.29, respectively. The free phosphate content were 4.12%, 4.79%, and 4.86%, and the phosphorus-to-aluminum molar ratios were 1.012, 0.972, and 0.944, respectively. The adsorption rates of aluminum adjuvant to TT were 92.7%, 87.8%, and 85.7%, and to 6A-TT were 93.8%, 91.6%, and 90.1%, respectively. The aluminum phosphate adjuvant, after autoclaving 1, 2, and 3 times, was able to adsorb 6A-TT and induce antibody production in mice, with geometric mean titers of 1 213, 985, and 1 056, respectively.Conclusions Increasing the number of autoclaving cycles leads to decrease in the isoelectric point and pH of the aluminum phosphate adjuvant, increase in free phosphate content, and reduction in the phosphorus-to-aluminum molar ratios and protein adsorption rate. It may also have an effect on the immunogenicity of 6A-TT pneumococcal polysaccharide antigen in mice.

Objective To evaluate the application effects of 3 types of sheet carriers for the production of Sabin strain inactivated poliovirus vaccine (sIPV) in order to provide basis for carrier selection in sIPV production process.Methods A 15 L basket bioreactor system was adopted to carry out Vero cell culture and prepare Sabin strain poliovirus liquid with 3 sheet carriers A, B, and C, respectively. The cell attachment rate of Vero cells inoculated on the sheet carriers within 0-3 h, the glucose metabolism characteristics during the 8-day culture cycle, the virus titer and D-antigen content of the virus harvest were investigated and compared.Results Cell attachment rates of carriers A, B, and C were all >90% at 3 h after inoculation. Cells in all 3 groups presented a typical S-shaped glucose consumption curve. The cumulative glucose consumptions of A and B were (191.1±7.6) g and (192.9±5.3) g, with no statistically significant difference (F=43.23, P=0.060), while both were statistically significantly higher than that of C at (153.1±4.4) g (F=43.23, P<0.001). D-antigen contents of A and B were (773.3±10.8) and (777.0±12.5) D-antigen unit (DU)/mL, and virus titers were (8.1±0.2), (8.2±0.1) lgCCID₅₀/mL,respectively, with no statistically significant difference (F=209.00, 27.30; P=0.946, 0.708). In contrast, the two indicators of group C at (598.3±14.2) DU/mL and (7.2±0.2) lgCCID₅₀/mL,were lower than those of group B with statistically significant differences (F=209.00,27.30; P<0.001).Conclusion Carriers A and B both exhibit clear advantages in maintaining the metabolic activity of Vero cells and promoting poliovirus replication.

Objective To validate the method of cell species identification (multiplex PCR) and apply it to the identification of cells from various species.Methods Cell genomic DNA was extracted by cell species identification detection kit (multiplex PCR), and the target gene was amplified by mixed primers. Multiplex PCR combined with agarose gel electrophoresis was used to identify species and detect cross-contamination according to the band size and number of amplified products, and the detection limit, specificity and durability to 5 different sources of cells were investigated.Results The detection limits of multiplex PCR method were 500 cells for Hep-2 cells (human), canine kidney cells, Vero cells (African green monkey) and L929 cells (murine), and 5 000 cells for Chinese hamster ovary cells. When each cell type was designed with cells from different sources for cross-contamination, both main cells and contaminant cells as low as 1‰ level were detected. When the genomic DNA extracts of 5 kinds of cells were stored at ﹣18 ℃ for 1, 3 and 7 d, the amplification results were consistent.Conclusion Multiplex PCR method has high sensitivity, strong specificity, rapidity and convenience, which is helpful to improve the quality control level of cell substrates for the production of biological products.

Objective To establish and verify the quantitative detection of endotoxin content in PEGylated uricase for injection by recombinant C factor assay.Methods The adjustment of gain value and reliability verification of standard curve were carried out by recombinant C factor assay. This method was verified for specificity, linearity, precision, accuracy, detection limit and durability. The interference test and detection of 3 batches of final products were carried out. The results were compared with those of gel method.Results When the instrument gain value was set to medium, the lg(Δ relative fluorescence units) was the closest to 3.5. The recombinant C factor did not react with β-glucan. The standard curve showed good linear relationship within the range of 0.005-5.000 endotoxin units (EU) /mL, and coefficients of determination were all ≥ 0.980.The relative standard deviation (RSD) of precision was ＜ 15.0%.The recovery rates of samples with different concentrations met the requirement of 50%-200%, and the RSD of the recovery rates was ＜ 15.0%.The detection limit was 0.005 EU/mL.The coefficients of determination with different gain values were all ≥ 0.980, and the RSD of the measured values was ＜ 15.0%. The interference test result showed that the recovery rate was 97.2% when the test sample was diluted 40 times and 0.5 EU/mL standard endotoxin was added. The test results of 3 batches of final products met the requirements of the standard limit and were consistent with the results of gel method.Conclusion The recombinant C factor assay has good specificity, linearity, precision, accuracy and durability, which can be used for quantitative detection of endotoxin in the final products of pegylated uricase for injection.

Objective To establish and validate a double-antibody sandwich ELISA for the detection of rabbit milk protein residues in recombinant protein drugs expressed by transgenic rabbit mammary gland bioreactor.Methods Polyclonal antibodies were obtained by immunizing sheep with non-transgenic rabbit milk, and a double-antibody sandwich ELISA for detecting rabbit milk protein residues was established. The method was validated for specificity, accuracy, precision and ruggedness, and its limit of quantitation, linearity and range were determined. The established method was applied to detect the residual amount of rabbit milk protein in recombinant human C1 esterase inhibitor expressed by transgenic rabbit mammary gland bioreactor.Results The average absorbance value difference of specificity test between 450 and 630 nm was ≤0.1, with good linearity at the concentration range of 5-800 ng/mL and limit of quantitation of 5 ng/mL. The recoveries of low, medium and high spiked samples were 111.0%, 117.5% and 110.4%, with relative standard deviations (RSDs) of 5.0%, 4.7% and 8.9%, and RSDs for intermediate precision of 7.9%, 8.5% and 7.6%, respectively. For the ruggedness test, RSDs of the determined values of low, medium and high spiked samples under 3 different experimental conditions were 8.0%, 4.7% and 7.6%, respectively.Conclusion A double-antibody sandwich ELISA for the detection of rabbit milk protein residues in products expressed by the transgenic rabbit mammary gland bioreactor is successfully established, which has good specificity, accuracy, precision, ruggedness and sensitivity.

Objective To isolate bacteria from cerebrospinal fluid of an infant suspected of epidemic cerebrospinal meningitis from Guangdong Province and to conduct genotype typing and drug resistance test.Methods The bacteria was isolated from cerebrospinal fluid and cultivated,and morphology observation, biochemical identification, serotype identification, genetic sequencing analysis and drug susceptibility test were conducted.Results The isolate showed greyish-white colonies on blood agar plates and appeared as non-capsulated, non-spore-forming, Gram-negative bacilli under the microscope. The results of biochemical reaction and mass spectrum identification of the isolated strain matched the characteristics of Salmonella and the serotype was 4,12:i:1,2. Genome sequencing implied that the strain was S.typhimurium, which belonged to the ST-19 group by multilocus sequence typing. The isolated S.typhimurium showed resistance to 10 antibiotics including cefotaxime, ampicillin, aztreonam, ciprofloxacin, etc.Conclusion S.typhimurium is isolated from the cerebrospinal fluid of an infant diagnosed as meningitis, and the isolate shows multi-drug resistance.

Immunometabolism is an emerging field that explores the interactions between immune cells and metabolism. In recent years, the role of immunometabolism in autoimmunity and autoimmune diseases has become a growing area of research, focusing on how cellular metabolism influences immune cell function and responses. It is currently understood that immune cell signaling and differentiation processes can determine immune cell behavior, control numerous metabolic pathways and thereby affect immune cell function and responses. This article primarily discusses the metabolic alterations in immune cells in systemic lupus erythematosus, type 1 diabetes and rheumatoid arthritis, elucidating how immunometabolism influences disease onset, exacerbation, or remission, and summarizes drugs that modulate immunometabolism to potentially control immune responses in autoimmune diseases and their research progress.

With the rapid development of microneedle technology, microneedle has gradually become a new vaccine delivery method, which has advantages such as minimally invasive, painless, self-administration, dose-saving, and enhanced immune response. The evaluation of immunogenicity is crucial for vaccine research and development. However, there is still no systematic summary of immunogenicity evaluation method of microneedle vaccines. This article summarizes the characteristics of immune responses to microneedle vaccines, systematically reviews and discusses various methods and key indicators for evaluating the immunogenicity of microneedle vaccines, as well as explores and prospects emerging immunogenicity evaluation methods in microneedle vaccines.

Recombinant adeno-associated virus (rAAV) vectors have become one of the preferred delivery vectors for gene therapy due to their safety and effectiveness. Currently the production of rAAV is mainly based on transient expression system in mammalian cells. However, the low production efficiency and high administration dose result in the high cost of gene therapy, which limits the scale-up of its application. As a large number of rAAV-based gene therapies enter the clinical trial and marketing stage, requirement on the production capacity of rAAV is increasingly urgent. Improving the synthesis efficiency of rAAV through in-depth understanding of transient expression system and rAAV biosynthesis characteristics of mammalian cells is the key to establish an efficient and economical rAAV production process based on animal cell culture. This paper reviews the fundamental characteristics and production methods of rAAV, focusing on the key factors affecting rAAV biosynthesis and host cell characteristics during the transient expression based on multiple plasmids, as well as the corresponding regulatory optimization strategies, which provide theoretical references for further development and optimization of rAAV expression system and production process to achieve efficient biomanufacturing of rAAV.