We produced and identified the monoclonal antibodies against Brucella melitensis U‐lipoprotein OMP19 .A DNA fragment coding omp19 of Brucella melitensis was amplified by PCR ,and inserted into the vector of pET‐30a(+ ) ,the result‐ant recombinant plasmid ,which we designated as pET‐30a(+ )/omp19 .We then transformed the plasmid into BL21(DE3) competent cells for the expression of the OMP19 protein .After induction with different concentrations of IPTG ,the colleted cells were analyzed by SDS‐PAGE ,and then OMP19 monoclonal antibodies were prepared through hybridoma technology . These mAbs were tested to reactivity to rOMP19 and nature membrance proteins (NMP) of Brucella melitensis by Western blot and IEST .We successfully constructed an expression vector of pET 30a(+ )/omp19 .An IPTG‐induced expression of the OMP19 protein (19 kDa in molecular weight) was demonstrated by SDS‐PAGE .The fusion protein existed in the form of solu‐ble ,and the OMP19 protein of high purity could be obtained by Ni‐NTA .Western blot assay showed that the refolded protein could be recognized by the anti‐serum against Brucella melitensis .Twenty‐three mAbs to OMP19 was produced in which 91 .30% were IgG1 ,twenty‐two (95 .65% ) mAbs could recognize nature OMP19 protein ,and eighteen (78 .26% ) mAbs could recognize NMP ,four mAbs could react with Brucella melitensis .The protein maintained good immunogenicity and twenty‐three mAbs were obtained ,which we believe provides a good protein candidate for the immunological research .

Objective To investigate the humoral and cellular immune responses in patients with acute brucellosis, and evaluate dynamic changes of regulatory T-lymphocytes (Foxp3+ Treg) in the peripheral blood of patients during treatment, in order to clarify the relationship between immunosuppression and the therapeutic effect in human brucellosis.Methods Sixty-five patients with brucellosis hospitalized at the Third Department of Infectious Diseases, Heilongjiang Agriculture and Reclamation Bureau General Hospital between July 2015 and November 2015 were included.Twenty-eight patients were treated with conventional therapy (group A: patients received 3 courses of treatment.Each lasted for 20 days with one-week interval), and 37 patients were treated with conventional therapy in combination with immunopotentiator (group B).Thirty healthy volunteers were enrolled as the controlled group.The ratio of CD3+CD4+ Foxp3+ Treg cells in the peripheral blood of brucellosis patients were measured by flow cytometry (FCM) at the end of each course of treatment.Data in accordance with normal distribution were described as mean±standard deviation.Comparison between two groups was done by two sample t test.Comparison among multiple groups was performed by analysis of variance and SNK test.Data that did not fit the normal distribution were analyzed by multiple-sample nonparametric test.Results After the first (20 d), second (50 d) and third course of treatment (80 d), the ratios of Foxp3+Treg in the peripheral blood of 65 acute brucellosis patients were 2.83%, 3.77% and 4.03%, respectively, which were all significantly higher than control group (1.69%;t=5.97, 9.05 and 5.66, respectively, all P<0.01).At the end of the first course of treatment, the ratios of Foxp3+Treg in group A and B showed no statistically difference (t=0.33, P>0.05), while those were both higher than control group (t=7.09 and 4.94, respectively;both P<0.01).At the end of the second course, the ratio of Foxp3+ Treg in group B was higher than group A (t=2.22, P<0.01), and both of them were higher than control group (t=10.79 and 7.25, respectively;both P<0.01).At the end of treatment, Foxp3+ Treg in group A was also significantly higher than the other two groups (t=6.02 and 6.45, respectively;both P<0.01).Conclusions In patients with acute brucellosis treated with the standard antibiosis treatment in combination with immunopotentiator, the ratio of Foxp3+Tregs significantly increases and maintains at a high level, which suggests that extra immunopotentiator may be not helpful for the treatment of brucellosis at the very early stage.

Objective To investigate the percentage of regulatory T cells (Treg) in peripheral blood lymphocytes of patients with chronic brucellosis and the percentage change before and after treatment of different regimens,and to analyze the influence of Treg cell-induced immunosuppression on the therapeutic effect of chronic stage brucellosis.Methods Using case-control study,35 patients with chronic brucellosis who were hospitalized in Heilongjiang General Hospital of Agriculture Bureau [28 males,7 females,aged (45.37 ± 20.16) years old] were selected as case group.According to the treatment regimen,they were divided into standard treatment group (15 cases) and immune enhancer group (20 cases),the treatment was 20 d;30 cases of in-hospital health examinations were selected [16 males and 14 females,aged (35.53 ± 11.38) years old] as control group.Peripheral blood sample of the subject was collected before and after the treatment,the Treg cells as a percentage in peripheral blood lymphocytes were detected by flow cytometry.And the percentage change of Treg cells of brucellosis patients who underwent different treatment regimens was analyzed.Results Before treatment,the percentage of Treg cells in peripheral blood lymphocytes of the control group,the standard therapy and the immune enhancer groups [(1.69 ± 0.38)％,(3.12 ± 0.86)％,(3.05 ± 1.07)％] was significantly different (F =25.89,P ＜ 0.05);compared with the control group,the percentage of Treg cells in the peripheral blood lymphocytes of the standard treatment group and the immune enhancer group increased (P ＜ 0.05);there was no significant difference between the standard treatment group and the immune enhancer group (P ＞ 0.05).After treatment,the percentage of Treg cells in peripheral blood lymphocytes of the control group,the standard therapy and the immune enhancer groups [(1.69 ± 0.38)％,(3.06 ± 0.76)％,(2.85 ± 0.89)％] was significantly different (F =30.84,P ＜ 0.05);compared with the control group,the percentage of Treg cells in the peripheral blood lymphocytes of the standard treatment group and the immune enhancer group increased (P ＜ 0.05);there was no significant difference between the standard treatment group and the immune enhancer group (P ＞ 0.05),and compared with the same group before the treatment,respectively,the differences were not statistically significant (P ＞ 0.05).Conclusions The percentages of Treg cells in peripheral blood lymphocytes of the chronic brucellosis patient are not significantly changed before and after different treatment regimens.It suggests that the immunesuppression induced by Treg cells may be one of the reasons why the host organism cannot effectively remove residual Brucella in the body,which leads to chronic infection.

Objective:To investigate the effect of recombinant lipoproteins of Brucella outer membrane protein 16, 19 (L16 and L19) on the expression of immune regulatory factors in human monocytic leukemia cell line (THP-1 cells). Methods:THP-1 cells activated with phorbol ester (PMA) were used as an in vitro experimental cell model, and a group design was used to co-culture L16, L19 and THP-1 cells (L16 stimulated group, L19 stimulated group), respectively. THP-1 cells activated with PMA were used as the control group. When co-cultured for 4 hours, immunofluorescence staining (IFS) and Western blotting were used to detect whether L16 and L19 entered the cells, respectively; when co-cultured for 12, 24 hours, real-time fluorescent quantitative PCR was used to measure the mRNA expression levels of interferon regulatory factor 1 (IRF-1) and trans activator protein of major histocompatibility complex class Ⅱ (CⅡTA); Western blotting was used to detect the protein expression levels of T cell immunoglobulin mucin-3 (Tim-3) and γ interferon receptor 1 (IFNGR1). Results:When co-cultured for 4 hours, L16 and L19 were observed entering THP-1 cells in the L16 stimulated group and L19 stimulated group, respectively. When co-cultured for 12 hours, the expression level of IRF-1 mRNA in the L16 stimulated group (0.16 ± 0.15) was significantly lower than that in the control group (1.00 ± 0.00, P < 0.05). When co-cultured for 24 hours, the expression level of CⅡTA mRNA in the L16 stimulated group (0.17 ± 0.10) was significantly lower than that in the control group (1.00 ± 0.00, P < 0.05). When co-cultured for 12 and 24 hours, there were no statistically significant differences in the expression levels of IRF-1 and CⅡTA mRNA between the L19 stimulated group and the control group ( P > 0.05). Western blotting results showed that there were statistically significant differences in the expression levels of INFGR1 and Tim-3 protein among the control group, L16 stimulated group, and L19 stimulated group after co-cultured for 12 and 24 hours ( F = 50.92, 6.80, 148.73, 156.57, P < 0.05). Among them, when co-cultured for 12 hours, the expression level of INFGR1 protein in the L16 and L19 stimulated groups were significantly lower than that in the control group, and the L19 stimulated group was higher than the L16 stimulated group ( P < 0.05), and the expression level of Tim-3 protein in the L19 stimulation group was higher than that in the control group ( P < 0.05). When co-cultured for 24 hours, the expression level of INFGR1 protein in the L16 and L19 stimulated groups were lower than that in the control group, and the L19 stimulated group was higher than that in the L16 stimulated group ( P < 0.05); and the expression level of Tim-3 protein in the L16 stimulated group was higher than that in the control group and L19 stimulated group ( P < 0.05). Conclusions:Brucella L16 can downregulate the expression levels of IRF-1 and CⅡTA mRNA in THP-1 cells. Both L16 and L19 can downregulate IFNGR1 and upregulate Tim-3 protein expression levels.

Objective:Using the monoclonal antibody to Brucella Omp31, flow cytometry (FCM) method for detecting Brucella antigens is established, and to analyze its potential value in clinical diagnosis. Methods:The supernatants of sonicated proteins (SSPs) from Brucella abortus (2308, 104M and S19), Brucella melitensis (M5-90), and Brucella suis (S2) were identified by Western blotting and enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (mAb) 5H3 to Brucella Omp31, which were prepared by breaking Brucella species with ultra-sonication. The recombinant eukaryotic plasmid (pcDNA3.1-Omp31) was constructed and transfected in 293FT cells, and the expression of Omp31 was detected by Western blotting. THP-1 cells were infected by Brucella melitensis M5-90 strain to simulate mononuclear phagocytes carrying with Brucella spp. To identify the ability of mAb 5H3, FCM for detecting intracellular Brucella was established, mAb 5H3 was labeled with fluorescein isothiocyanate (FITC-5H3) or P-phycoerythrin (PE-5H3), and then the transfected 293FT cells and THP-1 cells invaded by M5-90 strain were individually identified by FCM with FITC-5H3, and sensitivity of FITC-5H3 in FCM was tested. The PBMCs collected from brucellosis patients or normal blood donors were tested by FCM with double mAbs including PE-5H3 and FITC-CD14 to evaluate this method's feasibility in clinical practice. Results:MAb 5H3 was able to identify Brucella melitensis (M5-90) and Brucella suis (S2), as well as Brucella abortus (2308, 104M and S19) with Omp31 gene deletion. The mAb 5H3 labeled with FITC or PE was used for identifying Brucella antigen in various cells by FCM. The results revealed that the proportion of 293FT positive cells expressing Omp31 was about 59.3%, and the proportion of THP-1 positive cells infected by vaccine strain M5-90 was about 6.2%. In addition, the sensitivity of FCM with FITC-5H3 for the 293FT cells transfected with pcDNA3.1-Omp31 was about 4%. The FCM based on double mAbs staining of PE-5H3 and FITC-CD14 was preliminarily established. For brucellosis patients, the proportion of cells (1.93%) stained with the double mAbs in PBMCs was higher than that of normal blood donors (< 0.30%, negative) in FCM. Conclusions:A FCM assay is preliminary established basing on mAb 5H3 against Omp31 for detecting intracellular Brucella. Moreover, we have found that mAb 5H3 could recognize Brucella abortus originally lacking Omp31, which reduces the defect of Omp31 applied in all Brucella species detection. The development of this FCM assay provides a new strategy and usable reagents for brucellosis pathogens diagnosis.

Objective:This study is designed to investigate the toxicity of lipoprotein (L16) and non-lipoprotein (U16) of Brucella outer membrane protein (OMP) 16 on osteoblasts. Methods:Recombinant L16 and U16 proteins were prepared by using prokaryotic expression system of Escherichia coli ( E. coli) BL21 (DE3) and purified by Ni column. Using group design, mouse osteoblasts (MC3T3 cells) were co-incubated with L16 and U16, respectively. Brucella lipopolysaccharide (LPS) stimulus was used as the positive control, and cells without any stimulation were used as the negative control. Incubation time was 24 h. The activity of co-incubated MC3T3 cells were detected by CCK-8; the supernatant of cultured cells was collected and the release rate of lactate dehydrogenase (LDH) in the supernatant was detected by bioluminescence, and the virulence of L16 and U16 on MC3T3 cells was evaluated. Annexin Ⅴ-PE/7-AAD double staining flow cytometry was further used to analyze the apoptosis rate of MC3T3 cells, and the activation level of apoptosis executive protein Caspase-3 was detected by Western blotting (WB). Results:The activity of MC3T3 cells in L16 group [(56.16±1.63)%] was significantly lower than that in U16 and LPS groups [(97.02±1.44)%, (98.64±0.90)%, P < 0.01], the LDH release rate [(84.64±0.96)%] was significantly higher than that in U16 and LPS groups [(34.82±3.41)%, (26.75±1.95)%, P < 0.01]. Annexin Ⅴ-PE/7-AAD double staining results showed that the apoptosis rate was (46.45±2.19)% in L16 group, while the remaining groups were all less than 1%. WB results showed that activated Caspase-3 (cleaved-Caspase-3) existed in L16 stimulated cells, but not in U16 stimulated cells and LPS control cells. Conclusion:L16 can induce the apoptosis of osteoblasts and inhibit the proliferation of osteoblasts, but U16 has no obvious effect indicating that Brucella L16 with complete lipid structure is necessary for virulence effect.

【Objective】 To explore the relationship between clopidogrel responsiveness and CYP2C19 gene polymorphism by thromboelastography(TEG) after PCI in patients with coronary heart disease, and its guiding significance for the use of clopidogrel after PCI. 【Methods】 A total of 246 patients who underwent PCI surgery in our hospital from June 2018 to May 2021 and routinely took clopidogrel maintenance treatment after the operation were selected.The platelet inhibition rate of the patients was detected by TEG to obtain their response to clopidogrel.The CYP2C19 genotype was detected, and the relationship between the patient′s responsiveness to clopidogrel and the CYP2C19 genotype was analyzed. 【Results】 The CYP2C19 genotypes in 246 patients were fast metabolizer (n=95), intermediate metabolizer (n=104) and slow metabolizer (n=47), with the mean ADP inhibition rate(%) at 46.27±21.41, 40.99±25.53 and 24.77±21.68, respectively.They were divided into clopidogrel resistant group (n=98) and clopidogrel normal response group (n=148). The three groups of patients with different CYP2C19 genotypes had no statistically significant differences in gender composition, age and platelet count (P>0.05), while significant differences in hypertension, diabetes and hyperlipidemia(P<0.05). Further comparison of the responsiveness to clopidogrel by groups of genotypes showed that there was no statistical difference in the incidence of CR in patients with fast metabolizers and intermediate metabolizers (P>0.05), but they were all lower than those with slow metabolism patients (both P<0.05). The ROC curve was drawn by the ADP inhibition rate measured by TEG and the CYP2C19 genotype, and the area under the curve (AUC) was 0.730(>0.5). Statistically significant difference was noticed in the low responsiveness to clopidogrel by different CYP2C19 genotypes (P<0.05). The drug responsiveness of clopidogrel measured by TEG had strong correlation with the patient′s CYP2C19 genotype.When the ADP inhibition rate was the best cut-off value (27.10%), the sensitivity and specificity of CYP2C19 genotype being diagnosed as the slow metabolite type, was 73.37% and 70.21%, respectively. 【Conclusion】 The response of clopidogrel after PCI in patients with coronary heart disease is associated with CYP2C19 genotype polymorphism.The use of TEG to detect the ADP inhibition rate of patients has strong predictive effect on CYP2C19 genotype and has guiding significance on antiplatelet therapy in patients with coronary heart disease after PCI.