Objective: To construct a high effective GFP reporter plasmid for screening Streptoccus pneumoniae virulent genes by differential fluorescence induction. Methods: The SD-ENH-GFP region of plasmid pGreenTIR was cloned into a suicide plasmid pEVP3 which contains a cat gene encoding resistance protein to chloramphenicol,and a report plasmid pEVP3-SDGFP was constructed.To evaluate the function of this plasmid,a 500bp fragment of the pneumolysin gene (ply) of TIGR4 was coloned in the upstream of gfp and then was transformed into Streptococcus pneumoniae TIGR4. Results: The plasmid pEVP3-SDGFP could report the expression of ply both in vivo and in vitro,and was more effective than plasmid pEGFP-1 without SD and ENH sequence before gfp gene. Conclusion: Plasmid pEVP3-SDGFP can be used to construct the promoter-trap library which is needed in DFI.

Objective:To explore the expression of STAT3 and vascular endothelial growth factor (VEGF) in non-small cell lung cancer (NSCLC) and their relationship.Methods:SABC immunohistochemistry was used to detect the expression of STAT3 and VEGF in non-small tissues of 40 cases.Results:(1)The expression of STAT3 in NSCLCs had a obvious relation with the tumor cell differentiation and lymph node metastasis.No obvious difference in STAT3 expression was found among different tumor sizes of tumor types.(2)The expression of VEGF in NSCLCs had a significant relation with tumor size and lymph node metastasis.There was no significant difference between VEGF expression and tumor types and tumor cell differentiation.(3)There was significant positive correlation between the expression of VEGF and STAT3 in NSCLCs.Conclusion:STAT3 and VEGF may play an important role in the development of NSCLC.We identify that VEGF gene is regulated directly by STAT3 protein.

Objective:To obtain purified PspA produced by prokaryotic expression system.Methods:Template DNA was isolated from cultured Streptococcus pneumonia TIGR4.By gene recombination technology in vitro,sequence encoding PspA antigen epitope was cloned into PET-32(a) expression vector.After being confirmed by sequencing,expressed antigen protein was identified by SDS-PAGE and Western blot.Results:The DNA sequence analysis confirmed that the cloned PspA gene was according to GenBank data.The PspA fusion protein was proved by Western blot.Conclusion:A highly expressed recombinant PspA protein was successfully obtained.

To make sure the role of flagellin as mucosal adjuvants and the protective effect of streptococcus pneumonia infection when combined with DnaJ protein.Methods:Recombinant plasmid pET-28 ( a)/flagella was transferred to E.coli BL21(DE3).Over-expression of flagella was induced by IPTG and purification for animal study.All the C57BL/6 mice were randomly divided into three groups ,then respectively intranasally given the mixture of flagellin and DnaJ protein ( experimental group ) , DnaJ protein( control group 1 ) and the mixture of GST and DnaJ protein ( control group 2 ).The serum IgG and its subtype , cytokines secreted by mice spleen cells were detected by ELISA.At last all the C57BL/6 mice were intranasally immunized with Streptococcus pneumoniaD39.The protective effect by survival times were evaluated.Results: The mice of experimental group could secrete high level of serum IgG and cytokines IFN-γ,IL-4 and IL-17A.What more,the survival rate of mice in experimental group was 60%,a significant statistical difference with the control group.Conclusion:The flagellin as an adjuvant can reinforce the immune response of DnaJ protein and have better protection of resistance D 39 infection.We suggest that flagellin can be used as protein vaccine adjuvants.

Objective To evaluate whether immunization with △A146 Ply could confer protections against pneumococcal infections in murine models and to reveal the possible role of △A146 Ply-specific IgG in the protection elicited.MethodsBALB/c mice were immunized intraperitoneally with △A146 Ply or PBS plus alum.Fourteen days after the third immunization,mice were intranasally challenged with serotype 14 and 19F Streptococcus pneumoniae.Three days after inoculation,lungs were removed from mice and homogenized in PBS,followed by plated on red cell plates.Viable bacteria were counted after overnight incubation.As to the sepsis models,vaccinated mice were challenged intraperitoneally with different dosage bacteria of D39 and serotype 3 strain.The numbers of CFU log10 were compared by Mann Whitney U test and survival rates were analyzed using log-rank test.Passive protection was used to evaluate the role of △A146 Ply-specific IgG in the protection against otherwise lethal infection resulted from D39.Results ELISA analysis demonstrated higher titer specific antibody responses to △A146 Ply was produced after 3 times immunization.Mice boosted twice with △A146 Ply survived significantly longer than that for mice boosted once with △A146 Ply in Alum adjuvant,which were significantly longer than that of control group.Immunization with △A146 Ply was effective in reducing the numbers of pneumococcal strain 31614(serotype 14) and 31693(serotype 19F),which resulted in 50-and 20-fold decreases in bacterial load in the lungs respectively when compared to control protein-immunized mice.60% of vaccinated mice survived the infection with pneumococcal D39 of 1200 CFU.60% protection was achieved when mice intraperitoneally infected with D39 and received △A146 Ply-specific IgG,whereas no mice survived the infection when they were passively administered with △A146 Ply-specific IgG depleted antisera.Conclusion Immunization with △A146 Ply could confer protection against pneumococcal infections,and protection elicited was mediated by △A146 Ply-specific IgG.

According to the constructivism approach, instructors have to adapt to the role of fa-cilitators but not teachers. Whereas a teacher gives a didactic lecture that covers the subject matter , a fa-cilitator helps the learner to get to his or her own understanding of the content. In the former scenario the learner plays a passive role and in the latter scenario the learner plays an active role in the learning pro-cess. Under the guidance of this theory, a multi-dimension teaching model based on classroom teaching, network platform and innovate experiments has been established in the course of basis of clinical labora-tory. It has been found that this model is conducive to raising students' interests in learning and to culti-vating student's comprehensive quality.

Objective To lay the foundation for further exploration on parasitifer's defence reaction to pneumolysin through constructing ply gene-deletion strain of Streptococcus pneumoniae and researching on its virulence change. Methods A linker fragment with erm gene in middle and homologous upstream and downstream fragment of ply gene at both sides was prepared by long flanking homology-polymerase chain reaction(LFH-PCR). The linker fragment was transformed into Streptococcus pneumoniae. ply-deficient strain was then screened out from blood plate which contains erythromycin and identified by PCR. ply-deficient strain growth in vitro was observed and virulence change was observed through infecting mouse model. Results PCR results showed that ply gene was replaced completely by erm gene. The ply deficient strain was successfully constructed. The growth of single strain culture medium showed that ply genetic defect made no influence on bacterial's external growth. While in the mice nasal cavity infecting experiment, deficient strain enter into blood after 6 h from infecting which obviously slower than that did wild-type(2 h). And the number of bacteria at each point was much smaller than that of wild-type(P ＜0. 01 ). The mice peritonaeum infecting experiment showed that median lethal time of wild-type was 3 d, while that of deficient strain was 18 d(P＜0. 01). Conclusion It is a good way to completely substitute ply gene using LFH-PCR. ply deletion made no influence on baterial's growth in vitro, but it resulting in reduction of bacterial virulence in vivo.

OBJECTIVETo study whether previously intravenous injection of anisodamine can prevent endotoxemia of heat stroke of rats.

METHODSExperimental animals were randomly divided into two groups, their average artery pressure, heart rate, survival time, survival rate and rectal temperature were measured at an environmental temperature of 38 degrees C-40 degrees C and 50%-60% retative humidity. Blood samples for endotoxins analyses were taken both before and after heat-stress.

RESULTSDuring heat stress, the animals of rectal temperature of the experimental and control groups continuously increased and two hours later, separately to (42.7 +/- 0.6) degree C and (43.1 +/- 0.5) degree C, without statistic difference(P > 0.05), and to (44.6 +/- 0.4) degree C and (44.2 +/- 0.3) degree C prior to death, with statistic difference(P < 0.05). Before the experiment, the contents of endotoxins of portal vein blood were (45.7 +/- 5.2) pg/ml and (42.6 +/- 5.4) pg/ml, and that of systemic blood was (14.8 +/- 4.5) pg/ml and (13.9 +/- 7.2) pg/ml, without statistic difference(P > 0.05). Two hours later, the contents of portal vein blood separately increased to (122.2 +/- 16.7) pg/ml and (49.7 +/- 10.2) pg/ml, obviously higher than that before heat-stress(P < 0.01). And there were clear statistic difference between the two groups(P < 0.01). The changing tendency of the heart rhythm is almost the same in two groups, that is, first rose and then fell. But it is without statistic difference before and two hours later(P > 0.05): before heat-stress, the average artery pressures were (13.3 +/- 0.6) kPa and (13.6 +/- 0.5) kPa, without statistic difference(P > 0.05), and two hours later, were (9.6 +/- 0.5) kPa and (8.6 +/- 0.6) kPa, with obvious statistic difference(P < 0.01). The survival time of the animals were (166.5 +/- 16.9) min and (144.5 +/- 18.2) min with obvious statistic difference(P < 0.01), the survival rate of heat stressed rats in the experimental group were obviously higher than control group(P < 0.01 or P < 0.05).

CONCLUSIONSAnisodamine can prevent endotoxemia in rats suffering heat stroke.

Animals ; Blood Pressure ; Body Temperature ; Endotoxemia ; prevention & control ; Endotoxins ; blood ; Heat Stress Disorders ; drug therapy ; mortality ; physiopathology ; Hot Temperature ; Rats ; Solanaceous Alkaloids ; therapeutic use ; Survival Rate

To construct a three-dimensional (3D) model of histidine kinase (HK) YycG protein in Streptococcus pneumoniae and to investigate the interaction between YycG and its substrate ADP for the purpose of providing a theoretical basis for YycG selective inhibitor discovery, we constructed a 3D model of YycG protein by homology modeling, and assessed the reliability of the model using ProCheck and Profile_3D software. Besides, the active-site cavity of YycG and the residues key for substrate interaction were analyzed by Autodock4.0. Sequence alignment indicated that the YycG of S. pneumoniae was homologous to that of Thermotoga maritima. The constructed 3D model of YycG adopted a similar folding pattern to the template and the two matched well. The conservative amino acids in the substrate-binding pocket, such as Asn145, Asn149 and Lys152, as well as the hydrophobic residues at the bottom of the pocket played important role in binding and hydrolyzing substrate ADP. We have successfully constructed a reliable model of YycG protein. The model can be used as a starting point for designing antibacterial drugs.
Adenosine Diphosphate ; chemistry ; Amino Acid Sequence ; Bacterial Proteins ; genetics ; metabolism ; Histidine Kinase ; Models, Molecular ; Molecular Sequence Data ; Protein Kinases ; metabolism ; Protein Structure, Tertiary ; Sequence Homology, Amino Acid ; Streptococcus pneumoniae ; enzymology ; genetics ; Substrate Specificity