Objective Currently, there is no commercially available quantitative detection kit for the main Felis domestic allergen (Fel d 1) in China. To establish a rapid detection method for Fel d 1, this study aims to prepare monoclonal antibodies against Fel d 1 protein. Methods The codon preference of Escherichia coli was utilized to optimize and synthesize the Fel d 1 gene. The prokaryotic expression plasmid pET-28a-Fel d 1 was constructed and used to express and purify the recombinant Fel d 1 protein. Subsequently, the recombinant protein was immunized into BALB/c mice and monoclonal antibodies (mAbs) were prepared by the hybridoma technique. An indirect ELISA was established using the recombinant Fel d 1 as the coating antigen, and hybridoma cell lines were screened for positive clones. The specificity and antigenic epitopes of the mAbs were confirmed by Western blot analysis. Finally, the selected hybridoma cells were injected into the peritoneal cavities of BALB/c mice for large-scale monoclonal antibody production. Results The recombinant plasmid pET-28a-Fel d 1 was successfully constructed, and soluble Fel d 1 protein was obtained after optimizing the expression conditions. Western blot and antibody titer assays confirmed the successful isolation of two hybridoma cell lines, 7D11 and 5H4, which stably secreted mAbs specific to Fel d 1. Antibody characterization revealed that the 5H4 mAb was of the IgG2a subtype and could recognize the amino acid region 105-163 of Fel d 1, while the 7D11 mAb was the IgG1 subtype and could recognize the amino acid region 1-59. Conclusion The high-purity recombinant Fel d 1 protein produced in this study provides a promising alternative for clinical immunotherapy of cat allergies. Furthermore, the monoclonal antibody prepared in this experiment lays a material foundation for the in-depth study of the biological function of Fel d 1 and the development of ELISA detection.
Animals ; Antibodies, Monoclonal/biosynthesis* ; Mice, Inbred BALB C ; Cats ; Mice ; Allergens/genetics* ; Glycoproteins/genetics* ; Enzyme-Linked Immunosorbent Assay ; Hybridomas/immunology* ; Recombinant Proteins/genetics* ; Female ; Antibody Specificity

Aim To evaluate the time-course curve of expression of TRPC1 and vascular tone of pulmonary arteries(PAs)mediated by SOCE in chronic hypoxia pulmonary hyperte-nsion rats.Methods Both tension of PA rings and expression of TRPC1 were tested in CH exposure (1 0.0 % ±0.5 %partialpressure ofoxygen ) induced pulmonary hypertensive (PH)rats,and the time-course curve(detected respectively in CH 1 ,3,5, 7,1 4,21 d)was traced.Results ①CH could up-regulate the mean right ventricular pressure(mRVSP) ,which was increased significantly on 1 d,and reached the maximum on 7d;right ventricular weight index (RV-MI)began increase on 3d,and kept rising;②semi-quantitative reverse transcription polyme-rase chain reaction (RT-PCR)was performed to detect the expression of TRPC1 in PAs.The expression of TRPC1 increased significantly on 1 d,and reached the maxi-mum on 3d;③CH could up-regulate the vascular tone of PAs mediated by SOCE,which was increased signif-icantly on 3d,and reached the maximum on 7d.Con-clusions TRPC1 /SOCE increases significantly in the early days of CH,and the time-course curve of the two has correlation,which reflects the important role of the upregulation of TRPC1 /SOCE in the process of chronic hypoxia pulmonary hypertension.