Objective:To clone and express Mycobacterium tuberculosis(Mtb) Rv2460c gene(encoding ClpP2 protein),and evaluate the immunogenicity of its coding product. Methods: The recombinant plasmid of pET32a (+) vector-ClpP2 that H37Rv Rv2460c gene was cloned into the plasmid pET32a(+)vector,was transformed into E. coli BL21(DE3)and induced expression by IPTG,then purified by affinity chromatography. The recombinant protein was confirmed by SDS-PAGE and Western blot. The analysis of immunogenicity of Mtb ClpP2 and its epitope prediction were performed by bioinformatic methods. The antibody levels of polyclonal antibody titer against ClpP2 protein in rabbits and TB patients′ serum were detected by ELISA. Results: The recombinant ClpP2 protease was expressed as inclusion bodies in E. coli. The purity of purified protein was 93% by bandscan software analysis. The bioin-formatics analysis shows Mtb ClpP2 protein has multiple preponderant B cell and T cell epitopes. Rabbit antiserum titer was 1∶64 000;Serum anti-ClpP2 antibody levels in TB patients was higher than that in healthy control subjects. Conclusion:The recombinant ClpP2 protein was purified, and specific Rabbit anti-ClpP2 polyclonal antibody was prepared successfully. Experiment and bioinformatic information studies showed that Mtb ClpP2 protease has strong immunogenicity.

Objective:To investigate the antigenicity of ClpC 2 and the feasibility of polyclonal antibodies of ClpC 2 as detected antibody.Methods:rClpC2 was induced with IPTG.The rClpC2 was identified by SDS-PAGE and Western blot ,and purified by affinity chromatography ,with which rabbit were immunized and the specificity of rabbit antiserum was detected by Western blot , the titer of rabbit antiserum against ClpC2 was detected by double immunodiffusion and indirect enzyme-linked immunosorbent assay (ELISA).The antigenicity of the rClpC2 was detected by ELISA.The polyclonal antibodies of ClpC 2 were prepared to detect the ClpC 2 in clinical serum of TB patients by ELISA.Results:SDS-PAGE showed specific protein band with a relative molecular mass of 46 kD.The rClpC2 could bind with the antibody in the blood serum of the mouse immuned by MTB.By Bandscan analysis rClpC 2 accounted for about 58.7%of the total bacteria protein ,the purity of rClpC2 was 88.5% after purification.The ClpC2 of BCG could bind with the rabbit antiserum.The titer of antiserum were 1∶32 and 1∶320 000 by double immunodiffusion and ELISA detected respectively.ELISA results showed that clinical serum positive rate of rClpC 2 antigen was 46%in TB patients,the sensitivity of this protein was 46%,and the spe-cificity of this protein was 90%.ELISA results showed that the sensitivity of rabbit antiserum against ClpC 2 was 40%, and the specificity was 90%.Conclusion: Successfully expressed and purified rClpC 2 and high titer polyclonal antibody were successfully prepared,and these results will provide basements for further study on the biological functions of ClpC 2 and its candidate potentiality as serological diagnosis and drug-target and biological functions of antiserum against ClpC 2.

Objective:To elucidate the effect of hsa-microRNA-218(hsa-mir-218)on exogenous granulysin (GLS) expression in 293T cells.Methods:Total RNA was extracted from THP-1 cells induced by phorbol 12-myristate 13-acetatefor (PMA), and GLS gene was amplified by RT-PCR, and then cloned into pDsRed-Express-C1 to construct the GLS expression vector pDsRed-GLS.Then 293T cells were co-transfected with pDsRed-GLS and pGenesil-mir-218 (pGenesil-mir-control) and laser confocal microscopy was per-formed 36 h later to detect their co-expression .Total RNA and protein were extracted 48 h post transfection , and RT-PCR and Western blot were performed to detect the effect of hsa-mir-218 on exogenous GLS expression .Results:The GLS expression vector pDsRed-GLS was constructed successfully and laser confocal microscopy indicated that it was co -expressed with the interference vector .Compared with that of cells transfected with pGenesil-mir-control, Western blot showed a markedly decrease of GLS protein expression (50%) in the cells transfected with pGenesil-mir-218.However, GLS mRNA expression remained unchanged .Conclusion: hsa-mir-218 nega-tively regulates GLS expression at a post-transcriptional level , and this provides an experimental basis for future study of mechanism of GLS expression regulated by mir-218 .

Objective To understand the distribution of allergens in children with common allergic diseases in Guangzhou, and to provide evidence for the prevention and treatment of environmental allergens in children with different allergic diseases. Methods This study enrolled a total of 6 869 children with allergic diseases, including wheezing diseases and asthma (1 013 cases), allergic rhinitis (660 cases), allergic conjunctivitis (422 cases), eczema (2 762 cases), atopic dermatitis (831 cases) and urticaria (1 181 cases). Western blotting was used to detect the serum allergen type E specific antibodies (sIgE) of the patients. The results were statistically analyzed. Results The total positive rate of sIgE in 6 869 children was 74.01%. The positive rate of male children (75.02%) was higher than that of female children (72.25%). The positive rate of inhalation allergens (53.47%) was higher than that of food allergens (57.39%). House dust mites and milk were the most common inhalation allergens and food allergens, respectively. Eczema was the most common allergic disease in infants and toddlers. Allergic rhinitis and urticaria were common in school-age children. Conclusion The main allergens causing allergic diseases in children in Guangzhou were dust mites and milk. Different allergic diseases had its specific allergens. It is necessary to take targeted prevention measures accordingly.

AIM: To investigate the relationship between the levels of serum dehydroepiandrosterone(DHEA), erythropoietin(Epo), vasohibin-1(VASH-1)and diabetic retinopathy(DR)in patients with type 2 diabetes mellitus(T2DM). METHODS: Totally 185 T2DM patients(185 eyes)treated in our hospital from April 2021 to March 2024 were selected and divided into T2DM group(102 eyes)and DR group(83 eyes)based on whether retinal lesions occurred. DR patients were divided into nonproliferative DR group(NPDR, 47 cases, 47 eyes)and proliferative DR group(PDR, 36 cases, 36 eyes)based on the severity of their lesions. Fully automatic biochemical analyzer was used to detect serum biochemical indicators. ELISA was applied to detect serum levels of DHEA, Epo, and VASH-1. Pearson correlation was applied to analyze the correlation between serum DHEA, Epo, and VASH-1 levels and the severity of diabetic retinopathy. Logistic regression was applied to analyze the factors affecting the occurrence of DR. ROC curve was applied to analyze the diagnostic value of serum DHEA, Epo, and VASH-1 levels for T2DM combined with DR or PDR.RESULTS: Compared with the T2DM group, the DR group showed significantly increased DM duration, fasting blood glucose, uric acid, creatinine, and Epo, while DHEA and VASH-1 levels were significantly reduced(all P<0.05); Compared with the NPDR group, the PDR group showed a significant decrease in serum DHEA and VASH-1 levels, and a significant increase in Epo levels(all P<0.05); the levels of serum DHEA and VASH-1 were negatively correlated with the severity of DR, while the level of Epo was positively correlated with the severity of DR(all P<0.05); DHEA and VASH-1 were protective factors against DR, while Epo was a risk factor for DR(all P<0.05); the AUC of serum DHEA, Epo, and VASH-1 levels alone and in combination for the diagnosis of T2DM with DR were 0.804, 0.797, 0.805, and 0.903, respectively. The combined diagnostic value was higher than that of single diagnosis(all P<0.05); the AUC of serum DHEA, Epo, and VASH-1 levels alone and in combination for diagnosing PDR were 0.852, 0.850, 0.841, and 0.946, respectively. The value of combined diagnosis was significantly higher than that of individual diagnosis(all P<0.05).CONCLUSION:The levels of serum DHEA and VASH-1 in DR patients are clearly reduced, the level of Epo is clearly increased, and their levels are closely related to the severity of DR patients; therefore, combined detection has higher value for T2DM complicated with DR or PDR.