In the present study, the fusion gene lipL32/1-lipL41/2 from Leptospira interrogans was constructed by using PCR with linking primers, expressed in prokaryotic expression system. And the immuno-reactivity of its expressed product was determined. Meanwhile, SDS-PAGE and BioRad agarose image analyzer system was used to examine the expression output of the target recombinant protein rLipL32/1-LipL41/2, and the immune-reactivity of this recombinant protein was identified by Western blot assay. ELISA was used to detect the level of antibodies against the recombinant proteins in sera of patients with leptospirosis. It was demonstrated that the percentages of similarity in nucleotide and the putative amino acid sequences of the fusion gene lipL32/1-lipL41/2 with the corresponding sequences previously reported were 99. 9% and 98. 9%respectively. The expression output of the target recombinant protein was approximately 10% of the total bacterial proteins.Both the rabbit antisera against rLipL32/1 and rLip41/2 could recognize and combine to the recombinant fusion protein rLip32/1-Lip41/2 as well. The positive rates of antibodies in 228 leptospirosis patients with the recombinant proteins rLip32/1,rLip41/2 and rLip32/1-Lip41.2 were 97.4%, 78. 5% and 99. 1% respectively. The results of the present study leads to the conclusion that the fusion gene lipL32/1-lipL41/3 with its prokaryotic expression system is successively constructed and the expressed recombinant protein shows excellent immuno-reactivity, thus suggesting that it may be used as the antigenic candidate for the development of the leptospiral genus-specific engineering vaccine and for the preparation of diagnostic kits.

Objective To investigate the potential roles of pattern recognition receptor in the pathogenesis of asthma,the concentrations of serum nucleotide -binding oligomerization domain like receptors 1 (NOD1 )and Toll like receptor 1(TLR1)and Dectin and sCD14 protein were determined.Methods Blood samples were obtained from 45 cases of severe asthma(asthma group)during acute attack and convalescent period,the concentrations of serum NOD1,TLR1,Dectin and sCD14 protein were assessed using enzyme linked immunosorbent assay(ELISA),and compared with 30 healthy children(control group).Results The concentrations of NOD1,TLR1,Dectin and sCD14 protein were significantly higher in acute attack period of the asthma group[(65.53 ±19.95)ng/mL,(10.46 ±3.35)ng/mL, (80.38 ±19.51)ng/mL,(4.51 ±1.29)ng/mL]than those in the control group[(25.57 ±9.64)ng/mL,(5.54 ± 1.49)ng/mL,(47.37 ±15.16)ng/mL,(2.21 ±0.68)ng/mL](t =11.57,8.63,7.82,9.95,all P <0.01).Moreo-ver,the concentrations of serum NOD1,Dectin and sCD14 protein were dramatically decreased in convalescent period of the asthma group[(39.67 ±11.25)ng/mL,(60.04 ±16.76)ng/mL,(3.38 ±0.97)ng/mL]compared with acute asthma attack period(t =8.19,6.32,5.42,all P <0.01),while the concentration of serum TLR1 was not different between this two period (9.51 ±3.19)ng/mL(t =1.27,P >0.05).Furthermore,level of NOD1 was positively corre-lated with sCD14(r =0.49,P <0.01).Conclusion Levels of serum NOD1,TLR1,Dectin and sCD14 protein were increased in acute severe asthma,whereas,they were decreased in convalescent period.The elevation of them indicated that pattern recognition reception has synergistic function in the pathogenesis of asthma attack.

AIM:To construct a prokaryotic expression system of staphylococcal enterotoxin A(SEA)gene and determine the effects of the recombinant expression product rSEA in promoting lymphocyte proliferation and inhibiting tumor cell growth.METHODS:PCR was used to amplify entire SEA gene of S.aureus strain ATCC13565.The cloned SEA gene was sequenced after T-A cloning.SDS-PAGE was applied to measure the output of rSEA expressed by pET32a-SEA-E.coli BL21DE3.Ni-NTA affinity chromatography was performed to extract rSEA.Cytotoxicity of rSEA to Vero cells was detected using TCID_ 50 titration method and then the value of TCIC_ 50 was determined.MTT colorimetry was established to examine the effects of rSEA at different dosages on proliferation of mouse splenocytes and human peripheral blood mononuclear cells(PBMC)as well as on growth of HepG2 cells and HeLa cells in vitro.RESULTS:In comparison with the published corresponding sequences,similarities of the nucleotide and putative amino acid sequences of the cloned SEA gene were 100%.The output of rSEA was approximate 25% of the total bacterial proteins.rSEA had a cytotoxicity with TCIC_ 50 of 3.14 ?g to Vero cells.1.0-20.0 mg/L rSEA showed the significant effects of promoting proliferation of mouse splenocytes and human PBMC(P

Objective To investigate the effects of cisplatin on the expression of nucleotide － binding oligomerization domain－like receptor(NOD) 1 and 2 in human osteosarcoma SaOS－2 cell line,and to explore the mechanism of cisplatin in the treatment of human osteosarcoma. Methods CCK－8 assay,real－time quantitative reverse transcription polymerase chain reaction ( qRT － PCR ) and immumofluorescence methods were used to determine the growth survival rate and expression levels of NOD1 and NOD2 in osteosarcoma SaOS －2 cell line treated with cisplatin (0,5,10,20 μmol/L,named group S0,group S5,group S10,group S20,respectively) for 24, 48,72 hours. Results After treatment with cisplatin for 48 h or 72 h,the growth survival rates of SaOS－2 cells were significantly decreased in group S5 than those in group S0(65. 53% vs. 100. 00%;46. 43% vs. 100. 00%,χ2=8. 64,73. 97,all P＜0. 01). Moreover,after treatment with cisplatin for 24 h,48 h or 72 h,the growth survival rates of SaOS－2 cells were significantly decreased in group S10 or group S20 than those in group S0(80. 60% vs. 100. 00% , 42. 94 vs. 100. 00% ,27. 90% vs. 100. 00% ;62. 54% vs. 100. 00% ,33. 09% vs. 100. 00% ,22. 95% vs. 100. 00% , χ2=20. 99,79. 72,112. 50;45. 40,67. 56,125. 20,all P＜0. 01),and the growth survival rates were significantly lower in group S20 than those in group S5(62. 54% vs. 93. 78% ,33. 09% vs. 65. 53% ,22. 95% vs. 46. 43% ,χ2= 28. 47,21. 78,11. 71,all P ＜0. 01). The expression levels of NOD1 mRNA and NOD2 mRNA in group S5 were significantly increased at 48 h or 72 h than those at 24 h,and were higher than group S0 when treated with 5μmol/L cisplatin[(3. 64 ± 0. 44) vs. (4. 47 ± 1. 22) vs. (1. 79 ± 0. 44) vs (1. 00 ± 0. 00);(6. 88 ± 2. 79) vs. (6. 86 ± 2. 40) vs (2. 29 ± 0. 70) vs. (1. 00 ± 0. 00),F=29. 12,24. 11,all P＜0. 01]. And the expression levels of NOD1 protein had an increased tendency after 48 h or 72 h treatment with 5μmol/L cisplatin. Furthermore,the expression level of NOD1 mRNA was positively correlated with NOD2 mRNA(n=36,r=0. 92,P＜0. 01). Conclusion Cisplatin can elevate the function of osteosarcoma cell in a dose － and time － dependent manners, cisplatin may act as a efficient drug to cure osteosarcoma desease,which may be related to NOD1 and NOD2 signal pathway.

In order to increase antigenicity of H. pylori-specific antigens and decrease the cost of further industrial production, we used a special PCR with linking primers to construct a fusion gene containing H.pylori ureB and hpaA genes and E.coli ltB gene, and to costract its prokaryotic expression system pET42a-ltB-ureB-hpaA-E.coliBL21DE3. The sequencing result indicated the 100% nucleotide sequence homology of the constructed ltB-ureB-hpaA fusion gene compared to those of the original separated genes. Output of the target recombinant protein rLTB-UreB-HpaA was approximate 15% of the total bacterial proteins measured by SDS-PAGE. The rLTB-UreB-HpaA could induce the immunized rabbits to produce specific antibodies with immunodiffusion titer of 1∶8, and could combine to the commercial rabbit antibody against the whole cell of H.pylori as well as rabbit anti-UreB and anti-HpaA sera by using Western bolt assays. Using GM1-ELISA, the ability of rLTB-UreB-HpaA binding to bovine GM1 was confirmed.And rLTB-UreB-HpaA (200 μg per mouse) could prevent 100% of the immunized BaLb/C mice from H.pylori strain SS1 infection. The co-administration with 10 μg rLTB, the rUreB or rHpa could increase its protective rates in the immunized mice from 66.7% to 81.8% and 83.3%, respectively. All these data leads a conclusion that rLTB-UreB-HpaA is a great potential as a practical genetic engineering vaccine to prevent H.pylori infection.

Objective To investigate the effects of dexamethasone on expressions of epithelial neutrophil activating protein-78 (ENA-78) and transforming growth factor- beta 1 (TGF-β1) in neutrophil of asthma rats.Methods Thirty male SD rats were randomly divided into three groups on average, including asthma group, control group and dexamethasone treated group. In this experiment, the rat model of asthma were established by sentization and challenge with ovalbumin. Blood neutrophil were isolated and purified. The expression of ENA-78 was detected by flow cytometry. The expression of TGF-β1 was detected by immunohistochemical method in blood neutrophil and bronchial wall. Results Expression of ENA-78 in blood neutrophil in dexamethasone treated group(71.82 ±8. 87 mean fluorescence intensity)was lower than that in asthma group, but higher than that in control group(all P ＜0. 01). And expressions of TGF-β1 protein in dexamethasone treated group(0. 173 ± 0. 014,0. 202 ± 0. 019 optical density, respectively) was lower than that in asthma group(all P ＜0. 01) ,but higher than that in control group(all P ＜0. 01). There were significant positive correlation between ENA-78 expression at blood neutrophil and numbers of total inflammation cells in bronchoalveolar lavage fluid (n = 29, γ = 0. 762, P ＜ 0. 01). Conclusion The beneficial effect of glucocorticoid(dexamethasone) on airway inflammation in asthma rats could be at least in part due to their direct inhibitory effect on ENA-78 and TGF-β1 protein generation by neutrophil.

Objective To investigate possible role of 14 -3 -3 in the pathogenesis of asthma inflammation, the expression of 14 -3 -3 protein was observed in lung tissues of asthmatic rats.Methods Expressions of 14 -3 -3 protein was determined by immunohistochemisty method in lung tissues,and the relationship between 14 -3 -3 and asthma inflammation was analyzed.Results The location of positive expression of 14 -3 -3 protein was mainly at cytoplasm,while little at plasmalemma.The positive expression cell mostly was bronchial epithelial cell,others were lymphocytes,alveolar macrophages and vascular endothelial cells.On the other hand,the bronchial smooth muscle and vascular smooth muscle were negative expressed.Moreover,the expression level of 14 -3 -3 protein in asthma group [(0.353 ±0.023)absorbance]was significantly higher than that in the control group[(0.211 ±0.028 )absorbance] (t =10.969,P <0.01).Conclusion The results showed that the 14 -3 -3 protein was overexpressed in asthmatic lung tissue,it may play an important role in asthma inflammation through bronchial epithelial cells,lymphocytes, alveolar macrophages and vascular endothelial cell.

Objective To observe the expressions of grouth-related oncogen (GRO)α, epithelial neutrophil activating protein-78 (ENA-78) and neutrophil-activating peptide-2 (NAP-2) of rat asthma. And to investigate the role of neutrophil in the pathogenesis of asthma exacerbation. Methods In this experi-ment, the rat model of asthma were randomly divided into two groups on average, including asthma group and control group. Levels of ENA-78 at blood neutrophil were detected by flow cytometry method. The ex-pressions of GROα protein at bronchial wall and NAP-2 protein at blood neutrophil were detected by immuno-histochemieal method. Results Levels of GROα, ENA-78 and NAP-2 proteins in asthma group [0.138 ±0.009(A value), 97.65±13.99(MFI), 0.198±0.016(A value), respectively]were significantly higher than those in control group[0.077±0.010(A value), 50.79±8.66(MFI), 0.079±0.015(A value), re-spectively], all P < 0.01. Conclusion Levels of GROα, ENA-78 and NAP-2 were increased at rat asth-ma. They may be participate in inflammation of asthma exacerbation. Neutrophil may promote inflammatory cells influxing into airway wall via increasing synthesis of CXC chemotactic factors.

Objective To analyze and determine the efficient T- and B-combined (T/B) antigenic epitopes in Helicobacter pylori adhesin A. Methods Recombinant HpaA (rHpaA) was expressed for immunizing rabbit to generate antiserum. T- and B-cell epitopes in HpaA molecule were predicted by using bioinformatic technique. The segments to encode T/B combined epitope peptides were amplified by PCR and the phage display systems of T/B combined epitopes were subsequently constructed. PEG/NaCl precipitation method was applied to extract the recombinant phage PⅢ (rPⅢ) that displayed T/B combined epitopes. By using either commercial IgG against whole-cell of Helicobacter pylori or rHpaA antiserum as the primary antibody, the T/B combined epitopes displayed in rP Ⅲ s were screened and identified by Western blot and ELISA. MTT was applied to determine the proliferation of rHpaA-immunized mouse splenocytes after stimulation of the different recombinant rPⅢ proteins. Results In the HpaA molecule there were five T/B combined epitopes: HpaA10, HpaA37, HpaA79, HpaA116 and HpaA143. All the T/B combined epitopes were successfully displayed on the surface of PⅢ protein of phage M13. The results of Western blot, ELISA and MTT confirmed that HpaA116 was the predominant antigenic epitope, both HpaA37 and HpaA79 were the efficient antigenic epitopes. However, HpaA10 and HpaA143 were identified as ineffective antigenic epitopes. Conclusion The phage display systems of T/B combined epitope peptides of H. pylori adhesin A have been successfully generated in this study. HpaA37 and HpaA79, especially HpaA116 are the efficient T/B combined antigenic epitopes in HpaA of H. pylori.