HMGB-1, an ubiquitously expressed 25-KD nucleoprotein among mammals, belongs to the HMG family. Recent studies have identified that HMGB-1 is secreted by activated monocytes/macrophages via a non-classical ,vesicle-mediated secretory pathway. Extracellular HMGB-1 is an important proinflammatory cytokine. It participates in the pathogenesis of many diseases such as rheumatoid arthritis, sepsis , acute lung injury, and even leads animals death. Further studies of the mechanism and function of extracellular HMGB-1 may provide a novel strategy for the diagnosis and treatment of these diseases.

Objective:Using PCR to amplify filamentous fungi DNA fragments. Methods:Pour some liquid nitrogen to the hypha and grind them to powder. Then add some TE buffer and boil them for 15-20 minutes. Take a little supernatant as PCR template. After PCR do agarose gel electrophoresis. Collect the gel containing the objective fragment and add some TE. Take a little supernatant as PCR template (again.) After PCR, do agorose gel electrophoresis again. Results:We obtained a great deal of objective DNA. Conclusion:Amplifying filamentous fungi DNA fragment by this way, we can save time and money. We can also improve our work efficiently.

Objective To establish a multiplex polymerase chain reaction (PCR) assay for simultaneous identification of Shigella spp., Salmonella spp. and Vibrio cholerae. Methods Based on the gene sequences of invasion plasmid antigen H (ipaH) in Shigella spp., invasion plasmid antigen B(ipaB)in Salmonella spp. and enterotoxin extracellular secretion protein (EPSM) in V. cholerae, three pairs of primer were designed. Genomic DNA was extracted by the boiling method and multiplex PCR was performed with premix Taq in an ABI 2720 thermal cycle. The PCR-amplified products were then analyzed by using agarose gel electrophoresis. Results Under the optimized conditions, the assay yielded a 606-bp product from Shigella spp., a 314-bp product from Salmonella spp., and a 482-bp product from V. cholerae, respectively. When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons in different size were observed. Conclusions A rapid, specific and sensitive multiplex PCR system for simultaneous detection of Shigella spp., Salmonella spp. and V. cholerae has been established. The results suggest that the simultaneous amplification of several genes by multiplex PCR may provide an efficient and rapid diagnostic method for severe diarrhea.

This paper introduces such information of a urinary sediment diagnostic system and its data management system saving data directly from urine analyzer and microscope as its hardware organization and software design.With the performances of easy tooperate and low cost,the system can be applied toclinical urine diagnosis and togenerating standardized reports,whose data management system has high security and accuracy for saving data,strong anti-interference capability,reliable performance,noerror in proceeding and Chinese interface.

Objective: To clone human carboxypeptidase A1 and its active center gene as well as construct recombinant vector for expression before analysis their activities. Methods: CPA1 and CPAlactive center gene were amplified by RT-PCR from pancreas tissue, and then sequencing was carried out. The correct target genes were cloned into prokaryotic vector pGEX-4T-1 and transformed into E. coli BL21 before sequence analysis. After induced by IPTG, gene products were analyzed by SDS-PAGE. Target expressed proteins were denaturated, renaturated, purified and evaluated through MTT and agar colony form test. Results: Human carboxypeptidase Al and its active center gene were cloned successfully. New expected protein band of Mr 66,000 and 46,000 appeared on SDS-PAGE after inducement. Both of the expressed proteins have catalytic activity in vitro, but the activity of the latter is inferior when applied to tumor cells. Conclusions: Human carboxypeptidase A1 and its active center gene were cloned successfully. Their prokaryotic expression products were obtained too. The expressed proteins have catalytic activity in. vitro. A new prosperous beginning of further improvement for CPA1 therapy system has been established based on CPA1 and its active center gene in terms of ADEPT against prostate cancer to clinical application.

Objective To investigate the correlation between the results of HBsAg positive results among different detection systems,and provide reference for the analysis of clinical test results and the publication of the report.Methods The HBsAg positive serum specimens with the quantitation result lower than 80 COI were detected by Roche Cobas e602 electrochemical luminescence analyzer.All the specimens were also detected by Roche Modular e170 electrochemical luminescence analyzer.The differences of detecting results were compared and performed the linear correlation analysis.Results The experimental results of two detection systems are R2 =0.933.The positive coincidences of Group A,B,C,D and E were 60.60％,92.72％,96.66％,96.66％ and 100％,respectively.The positive coincidence of the males was 87.66％,while the positive coincidence of the females was 70.96 ％.The positive coincidence of the males was significantly higher than the females (P＜0.05).Conclusion The HBsAg detecting results of Roche Cobas e602 and Roche Modular e170 electrochemical luminescence analyzer had high correlation.The results higher than 10 COI had 100％ positive coincidence rate,however the results between 1 and 10 COI were not.The result between 1 and 10 COI may lead to controversial results,suggestions for further checks.

Objective To analyze the expression of urine exosomal miR-375 in prostate tumors and investigate its clinical utility.Methods A total of 45 patients with PCa,24 with benign prostate hyperplasia (BPH) and 24 healthy individuals were enrolled into this study.Exosomes were isolated from the urine of PCa,BPH and healthy individuals and the total RNA was extracted from the exosomes.The exosomal miR-375 expression was assessed by quantitative real-time PCR and analyzed with the comparative quantification cycle method (2-△△CT).We performed comprehensive biostatistical analyses to explore the clinical value of miR-375 in prostate cancer.Results The urine exosomal miR-375 expression was significantly downregulated in the patients with PCa compared with BPH and the healthy controls (P ＜ 0.01).No statistically significant difference of the urine exosomal miR-375 expression levels between the patients with BPH and healthy individuals was observed (P ＞ 0.05).The urine exosomal miR-375 expression level was also found to be associated with clinical stage and bone metastasis status of the patients with PCa (P ＜0.05),and with the increase of Clinical stage.The expression level of miR-375 decreased.No significant relationship was detected between miR-375 level and the patient's age,gleason score and serum prostate-specific antigen level (P ＞ 0.05).Receiver operator characteristic analyses demonstrated that the urine exosomal miR-375 expression could better differentiate PCa from BPH patients:AUC 0.715 (95％ CI:0.589-0.842) vs PSA AUC 0.632 (95％ CI:0.492-0.771) (P＜0.01).Conclusion The urine exosomal miR-375 could serve as a non-invasive biomarker for the diagnosis of PCa.

Objective Establishment and development of a novel Single-Nucleotide-Polymorphism TaqMan Real-Time PCR assay for rapid detection of rs12979860 that predicts HCV therapy response.Methods Human genomic DNA were extracted from solid tissues,secretion and plasma before allelic discrimination.With the property of minor groove binding protein (MGB) binding to minor groove of DNA with strong specificity and affinity,primers and MGB probes were particularly designed for differentiation of human genomic frequencies.MGB probe-based real-time PCR was established to increase allelic discrimination using two probes that only differ in one nucleotide of IL28B rs12979860.The specificity was evaluated by fluorescence signal emissions which were selected from two signal channels.And DNA sequencing was used to confirm the genomic polymorphisms.Results TaqMan probe-based SNP real-time PCR increased allelic discrimination using two probes that only differ by one nucleotide of amplicon,which indicated this assay was easily performed regardless of genomic DNA concentration and quality,minimizes sources of error.The sensitivity was as low as 1.5 ng/μl,the amplification efficacy was 97.6％.The genotype frequencies of CC,CT were remarkably different between Caucasian and Mongolian.The dominated genotype of Caucasian was CT,while most Mongolian was carried CC (26/40 vs.40/50,x-2 =18.75,P value ＜ 0.05).However,the genotype between two population showed no relationship with their virological clearance clinically (P value ＞ 0.05).Conclusions This TaqMan MGB assay shows highly specificity,which has potential as a routine diagnostic test for the detection of rs12979860 from various types of samples.This robust assay would be widely used clinically to predict patients' response before anti-HCV treatment in personalized medicine.

Objective To construct fusion gene and prokaryofie expression plasmid encoding the Rv1884c and Rv0867c genes in Mycobacterium tuberculosis (M.tb).The fusion protein wsg expressed efficienfly in E.coli cells.Methods The Rv1884c and Rv0867c genes were amplified by polymerase chain reactions (PCR) with specific primers from genomic DNA of M.tb H37Rv strain,and cloned into pGEX-4Tland pUC19 vectors.Rv1884c and Rv0867c were subcloned into the expression vector pGEX-4T-1 and pPRO-EXHT followed by DNA sequencing.The plasmids were transformed into E.coli DH5α and induced to produce GST-fused Rv1884c and His-fused Rv0867c fusion protein.The protein molecular weight and expression format was analyzed by SDS-PAGE.Results The recombinant expressive vectors pGEX-4T-1Rv1884c and pPRO-EXHT-Rv0867c were constructed.The DH5α strains of E.coli with recombinant plasmid showed high level of Rv1884c and Rv0867c gene expressions after IPTG induction.The SDS-PAGE showed that the plasmids expressed Rv1884c and Rv0867c fusion proteins with molecule weight of 45 000 and 80 000.The recombinant protein accounted for 18.3%and 23.7%of total bacteria protein.The expressed proteins could be purified via GSTrao FF and Ni2+-NTA system kits in denatured condition.Conclusions Mycobacterium tuberculosis Rv1884e and Rv0867c genes have been cloned and expressed Successfully in E.coli DH5α.The results lay a basis for further study of fast culfivation in Mycobacterium tuberculosis and investigation of their activities and functions.

Objective To investigate the immunological properties of Rv1009 domain. Methods BALB/c mice were immunized with Rv1009 domain three times at 2-week interval. ELISA was used to detect the antiRv1009 domain antibody titer in the sera of immunized mice sera. The spleen lymphocytes of the immunized mice were separated and the stimulation index (SI) was measured by MTT colorimetry. Levels of secreted IFN-γ, IL-10 and IL-12 upon specific antigen stimulation were detected by ELISA. The BALB/c mice immunized with Rv1009 domain were intravenously infected with MTB H37Rv. Four weeks after the final injection, the number of CFU in spleens was determined. Results The titer of the specific antibody in sera of the immunized BALB/c mice was 1:12 800. The SI of Rv1009 domain immunized group (2. 40±0. 18) was significantly higher than that of saline immunized group (0.90±0.21). The IFN-γ,IL-10 and IL-12 levels in culture supematant of spleen lymphecytes from the fusion proteins immunized mice was (1 432±30) ng/L, (503±11) ng/L and (311±11) ng/L respectively, significant different from that of saline immunized group[(256±20) ng/L, (76±6) ng/L and(56±8) ng/L,P<0.01]. Four weeks after the final injection,compared with normal saline immunized mice (6.64±0.13), dramatic reduction in MTB replication was observed in the spleen (4.86±0.14) from BALB/c mice immunized with fusion proteins following a subsequent MTB H37Rv challenge, but the protection efficacy of mice immunized with Rv1009 domain was not as good as that of BCG vaccination group (3.81±0.16). Conclusion Rv1009 domain can be used as a candidate for the new TB vaccine.