Objective:The envelope protein Domain I(DI) of type II mutated dengue virus from a DHF patient (B-E) was expressed,refolded and purified.Its immunogenicity was also identified.Methods:The 1-476 bp of B-E was cloned into pET28a(+),and it was transformed into E.coli after digestion and sequencing.The inclusion body was denatured and refolded after inducing with IPTG.Polyclone antibody was obtained by immunizing C57BL/6 and BALB/c mice with purified B-E,and identified with Western blot and ELISA.Results:The cDNA of B-E gene was inserted into pET28a(+) vector and transformed into Rosetta(DE3).The protein of B-E was mainly in inclusion body and the molecular weight was 20 kD as predicted.Western blot was used to identify the recommend protein with anti-his antibody.The soluble recombination protein was purified after denaturation and refolding.The polyclonal antibody was obtained by immunizing BALB/c and C57BL/6 mice and could be used in Western blot and ELISA assay.The titer of polyclonal antibody from C57BL/6 was 1:12 800 through ELISA assay,and titer of polyclonal antibody from BALB/c was 1:500 through Western blot.Conclusion:All the results suggests that B-E is immunogenic in BALB/c and C57BL/6 mice.

Objective:BALB/C mice model infected with different Dengue Type 2 virus (DV 2) strains was evaluated and dynamic patterns of humoral immune responses caused by the various viral strains were investigated.Methods:The BALB/C mice were multisubcutaneouly injected by different DV 2 strains. The dynamic changes of the anti-DV 2 IgM/IgG antibody were observed in the infected BALB/C mice by the indrect ELISA and the viremia was observed.Results:The productions of specific antibodies in the infected BALB/C mice after primary and secondary injection with different DV 2 strains showed some difference. Howerer, BALB/C mice infected by the DV 2 B strain from a DHF patient showed the prolonged viremia.Conclusion:The dynamic curve of specific antibody and the viremia induced by various DV 2 strain (DV 2 NGC strain.V.DV 2 B strain) were different in some existence.

Objective:BALB/C mice model infected with DV2 NGC strain was evaluated and dynamic patterns of cytokines were investigated.Methods:The dynamic changes of cytokines were observed in the infected BALB/C mice model by indirect ELISA was observed.Results:The dynamic levels of IL-4、IFN-? in the plasma of BALB/C mice after primary and secondary infected DV2 NGC strain were different.The results showed that the levels of IL-4 were significantly increasing in the early primary infected BALB/C mice while that of IFN-? were lower;In secondary infection ,IL-4 reached a peak on the first day( 4294 668 ? 349 038 pg/ml) and then the levels of IFN-? were higher on the forth and eleventh day.The concentrations of the cytokines produced infectious animals had closely relationship with the doses of DV2.Conclusion:Infection eliciting a dominant humonral immune response induced a high expression of Th2-related cytokines,whereas those show some appearance of Th1 cytokines.Th1 response,as described late for the disease infected with DV2 NGC strain,played a role while it inhibited Th2 response to some extend.Thus,it was responsible to recovery.Th cell and the cytokines were critic to the DV infective diseases.

Objective:To reveal the dynamic characteristics of the intracellular complement mRNA from mouse macrophage ANA-1 treated with LPS or IL-4. Methods:The polarization models of macrophage ANA-1 were established by treating with LPS(1μg/ml) and IL-4(20 ng/ml),respectively. After treating at 3,8,12 and 24 h,the total RNA were abstracted by Trizol lysis methods . The macrophage polarization were estimated by the expression of IL-1β, CCL2 and Arg-1 mRNA detected by Real-time fluorescent quantitative PCR. The intracellular complement C1q, C3, CfB and CRIg mRNA were quantitatively analyzed. Results: The mouse macrophage ANA-1 cells treated with LPS was polarized to M1 since the levels of IL-1β and CCL2 mRNA were up-regulated significantly,in which their 2-△△Ct value were up to 297. 0±31. 0 and 19. 9±3. 3 respectively at 12 h. On the other hand,the ANA-1 cells treated with IL-4 was polarized to M2 because the level of Arg-1 mRNA was obviously higher( the 2-△△Ct value of Arg-1 mRNA was up to 27.3±9.1 at 24 h)(P<0.05).The intracellular complement C1q,C3,CfB and CRIg mRNAs all were up-regulated in different polarized macrophages. The intracellular C1q and C3 mRNA in polarized M2 were significantly higher,in which the peak value of C1q and C3 were to 94. 9±12. 9 and 11. 3±2. 4 at 12 h,respectively(P<0. 05). Reversely,the CfB mRNA in polarized M1 increased obviously,in which its 2-△△Ct was to 61. 4±6. 2 at 12 h. In addition,the CRIg mRNA in both groups was only up-regulated at 24 h,in which the 2-△△Ct value was 6. 5±1. 8 in M1 and 10. 8±3. 2 in M2(P<0. 05). Conclusion: The macrophage ANA-1 cell polarization models were successfully established by treated with LPS or IL-4. The intracellular complement C1q,C3 and CRIg mRNA in polarized M2 were transcripted more than in M1. But the intracellular CfB mRNA in polarized M1 was up-regulated significantly. These results suggested that the dynamic characteristic of complement components in different polarized macrophage would be correlated with its fun-tions.

Objective To study on the expression of the eukaryotic recombinant vector carrying HlyX gene of Leptospira serovar Lai in mammalian cell and explore the humoral immune response in BALB/c mice immunized with the recombinant plasmid. Methods The HlyX gene was amplified from Leptospira serovar Lai genomic DNA by PCR and inserted into pcDNA3.1 vector. After transformed into E. coli DH5α,the recombinant plasmid was assayed for identification by PCR analysis,restriction nuclease enzyme digestion and sequencing. The recombinant plasmid was transfected into COS-7 cells,then RT-PCR and Western blot were performed to test the expression of the target gene. The recombinant plasmid was injected intramuscularly into BALB/c mice for three times at intervals of two weeks,and the antibody titer was measured by ELISA. Results PCR showed the full length HlyX gene was about 1100 bp. PCR analysis,restriction nuclease enzyme digestion and sequencing indicated the recombinant vector was constructed successfully. After the plasmid Was transfected into COS-7 cells,a fragment about 1100 bp was found by RT-PCR and a specific band relative molecular mass(Mr)about 40×103,which was consistent with the expected size of the target proteins was showed by Western blot. ELISA showed the antibody titer in BALB/c mice immunized by the HlyX gene of Leptospira serovar Lai can elicit high-titer antibody in BALB/c mice,which has laid the foundation for the application of the DNA vaccine.

Objective:To observe the effect of different concentration HMGB1 on the secretion of TNF-αand NO from Ana-1 infected with DEN2 and virus copy.Methods:DEN2 were proliferated and identified by conventional methods.The adherence of DEN2 to Ana-1 was observed by direct immunofluorescence and RT-PCR.The level of virus mRNA were detected by qRT-PCR.The concentration of TNF-αwas detected by ELISA.The concentration of NO was detected with Griess reagent.Results:Ana-1 was able to adhered for DEN2.Compared with DEN group,the inhibition ratio(%) of the level of virus mRNA in D-HMGB1-1 group,D-HMGB1-10 group,D-HMGB1-100 group,D-HMGB1-1000 group was 41.53 ±2.12,55.30 ±1.59,74.75 ±1.12,86.35 ±1.42.Compared with DEN group,the level of TNF-αand NO decreased in D-HMGB1 groups(P<0.05).Conclusion:HMGB1 can be effectively regulated of Ana-1 secreted inflammation factor of infected with DEN2,and inhibited DEN2 replication.

Objective:To investigate the impact of exogenous lactate on the replication of dengue virus type 2 (DENV-2) in Raw264.7 cells, mouse bone marrow-derived macrophages (BMDMs) and THP-1 cells and explore its association with cell activation.Methods:BMDMs from BALB/c mouse bone marrow were prepared and evaluated by flow cytometry to detect the proportion of F4/80 + CD11b + cells. Glucose transporter type 1 (GLUT1), hexokinase 2 (HK2), and monocarboxylate transporters 4 (MCT4) expression at mRNA level in BMDMs at different time points after DENV-2 infection were measured by qRT-PCR. The content of lactate in the culture supernatants was quantified via colorimetric assay. CCK-8 assay was used to evaluate the impacts of different concentrations of lactate on the viability of Raw264.7 cells, BMDMs, and THP-1 cells. qRT-PCR was used to detect the expression of DENV-2 E gene, TGF-β, CD86, retinoic acid-inducible gene Ⅰ (RIG-Ⅰ), IFN-β, interferon-stimulated gene 15 (ISG15), and ISG56 at mRNA level in cells infected with DENV-2 at different MOIs in the presence of different concentrations of lactate. Meanwhile, flow cytometry was used to analyze the expression of CD86 and CD206. Results:The percentage of BMDMs was (87.53±1.66)%. GLUT1 expression at mRNA level exhibited a decrease in BMDMs at 24 h after DENV-2 (MOI=3) infection following a transient increase at 12 h ( P<0.05), while HK2 expression at mRNA level was higher that than in blank control and inactivated DENV-2 infection groups at 12, 24, and 36 h ( P<0.01). Besides, there was an increase in both MCT4 mRNA level and the content of lactate in culture supernatants at 24 h after DENV-2 (MOI=1.5) infection ( P<0.05). The viability of the three types of cells remained above 80% when the concentration of lactate was 31.25 mmol/L. Lactate at the concentration of 35 mmol/L increased the expression of the DENV E gene at mRNA level in DENV-2-infected BMDMs at MOI=1 or MOI=2 ( P<0.05). Besides, it promoted the expression of DENV E gene at mRNA level in Raw264.7 and THP-1 cells ( P<0.001) as well as the expression of CD163, TGF-β, RIG-Ⅰ, IFN-β, ISG15 and ISG56 at mRNA level in BMDMs at MOI=1.5, but inhibited the expression of CD86 at mRNA level in BMDMs ( P<0.05). It also up-regulated CD206 protein expression ( P<0.01) and down-regulated CD86 protein expression ( P>0.05) in BMDMs. Conclusions:Exogenous lactate enhances DENV-2 replication in both human- and murine-derived macrophages and that might correlate with M2 macrophage polarization.

Objective To analyze the differences in the expression of specific antibodies targe-ting different human immunodeficiency virus(HIV)antigens among patients at different clinical sta-ges in Qiandongnan Prefecture of Guizhou Province,and to explore their association with the clinical staging of acquired immunodeficiency syndrome.Methods A total of 307 HIV-positive blood sam-ples from Qiandongnan of Guizhou Province were selected for specific HIV antibody immunoblotting assays.CD3+CD4+T cell counts andHIV viral load nucleic acid testing were performed on the blood samples.Multivariate regression analysis was conducted on relevant indicators during HIV in-fection and AIDS stages.Results Among the 307 HIV-infected individuals,218 were male and 89 were female,with a mean age of(48.53±16.03)years.The composition ratios of specific antibod-ies gp160,gp120,gp41,p66,p51,p31,p55,p24 and p17 were 98.7％,90.9％,92.2％,74.3％,66.4％,86.6％,3.9％,97.4％and 73.9％,respectively.Multivariate binary Logistic regression model analysis showed that expression of p24-specific antibodies were more likely to be judged as influencing factors in the infection stage(OR=0.158,95％CI,0.032 to 0.768,P＜0.05).There was a certain correlation between p24-specific antibodies and the clinical staging of HIV infection(OR=0.217,95％CI,0.005 to 0.944,P＜0.05).Using the ratio of CD3+CD4+T cell count to viral load when p24 was specifically expressed as the test variable,and the clinical stage as the state variable(AIDS stage=1,infection stage=2),a receiver operating characteristic(ROC)curve was plotted.The area under the curve was 0.653(95％CI,0.529 to 0.774,P＜0.05).Conclusion Differential expression of p24-specific antibodies may indicate that patients are in the infection stage and could potentially serve as an early warning indicator of immune function for HIV-infected individuals.

Objective To analyze the differences in the expression of specific antibodies targe-ting different human immunodeficiency virus(HIV)antigens among patients at different clinical sta-ges in Qiandongnan Prefecture of Guizhou Province,and to explore their association with the clinical staging of acquired immunodeficiency syndrome.Methods A total of 307 HIV-positive blood sam-ples from Qiandongnan of Guizhou Province were selected for specific HIV antibody immunoblotting assays.CD3+CD4+T cell counts andHIV viral load nucleic acid testing were performed on the blood samples.Multivariate regression analysis was conducted on relevant indicators during HIV in-fection and AIDS stages.Results Among the 307 HIV-infected individuals,218 were male and 89 were female,with a mean age of(48.53±16.03)years.The composition ratios of specific antibod-ies gp160,gp120,gp41,p66,p51,p31,p55,p24 and p17 were 98.7％,90.9％,92.2％,74.3％,66.4％,86.6％,3.9％,97.4％and 73.9％,respectively.Multivariate binary Logistic regression model analysis showed that expression of p24-specific antibodies were more likely to be judged as influencing factors in the infection stage(OR=0.158,95％CI,0.032 to 0.768,P＜0.05).There was a certain correlation between p24-specific antibodies and the clinical staging of HIV infection(OR=0.217,95％CI,0.005 to 0.944,P＜0.05).Using the ratio of CD3+CD4+T cell count to viral load when p24 was specifically expressed as the test variable,and the clinical stage as the state variable(AIDS stage=1,infection stage=2),a receiver operating characteristic(ROC)curve was plotted.The area under the curve was 0.653(95％CI,0.529 to 0.774,P＜0.05).Conclusion Differential expression of p24-specific antibodies may indicate that patients are in the infection stage and could potentially serve as an early warning indicator of immune function for HIV-infected individuals.