Objective To develop a method of loop-mediated isothermal amplification(LAMP) to Staphyloccocus aureus rapidly, specifically, sensitively and simply suited for the primary health agency. Methods According to conserved nucleotide of Staphyloccocus aureus and principle of LAMP, we designed a set of LAMP primers and set up an LAMP reaction system. We evaluated the specificity, sensitivity and re-peatability of the method. In addition,we evaluated the linearity between initial template copies 1g value and reaction time (the time when the fluorescent value is 1×10~4). Results The optimal assay showed that it was no cross-reaction with other closely related members of pathogens, and was 10 times more sensitive than PCR. The coefficient of variance between tests was less than 5% ,and the kinetics curves showed a good line-arity between initial template copies lg value and reaction time(r~2=0. 9501). The detection activity could be finished within 1 h with the sensitivity of LAMP was 100% and the specificity was 94.4%, and the accuracy was 96.6%. Conclusion These findings demonstrated that the LAMP had the potential clinical application for detection and differentiation of Staphyloccocus aureus in the public health agency for its sensitive, specific and simple feature.

Objective To detect the msr gene which confers resistance to erythromycin, and ana-lyze its distributing difference between the two biovars of Ureaplasma urealyticum. Methods Broth dilution method was used to determine the minimum inhibitory concentrations (MIC) to erythromycin among 72 U. urealyticum clinical isolates. The msrA, msrB, msrC and msrD genes detection and biotyping of U. urea-lyticum were conducted using PCR. Results The MICs of 72 U. urealyticum isolates to erythromycin ranged from ≤0. 125 μg/ml to ≥128 μg/ml. MIC_(50) was 32 μg/ml and MIC_(50) was ≥128 μg/ml. Biotyping showed that biovar Parvo had 51 strains (51/72, 70.83%) and biovar T960 had 21 (21/72, 29.17%) strains.The msrA, msrB, msrC and msrD genes were obtained in 1, 12, 0 and 24 strains, respectively, with five strains carrying the msrB and msrD genes, and one strain carrying the msrA, msrB and msrD genes. There was no resistance difference to erythromycin between the two biovars when the MIC≥8 μg/ml was considered resistance to eryt hromycin. But the msrB gene was predominantly detected in biovar T960. Conclusion U. urealyticum clinical isolates harbeur the msrA, msrB and msrD genes, and the predominantly detected msrB gene is of biovar T960.

Objective To search for the neutralizing epitopos on hepatitis E virus (HEV) capsid besides the known neutralizing epitope (aa459-606). Methods By analysis of several strains of monoclonal antibodies against HEV capsid and their recognized epitopes, the neutralizing activity of epitope (aa394-458) at N-terminus was compared with that of an immunodominant neutralizing epitope (an459-606). Re-suits The research showed a novel potential neutralizing epitope in aa423-437 of HEV ORF2 though detec-ting and comparing the characteristics of several antibodies and corresponding determinations. The epitope is a linear non-immunodominant epitope which is different from the other neutralizing epitope in aa459-606.And the amino acids sequence of this novel epitope is conservative. Conclusion ORF2 aa423-437 is a no-vel potential neutralizing leaner epitope of HEV. It is believed that the present work adds fundamental knowl-edge to our understanding of HEV capsid domain and contributes to the prevention and control of this dis-ease.

Objective To prepare VP1 protein vaccine of Coxsackievirus A16(CA16) and evalu-ate immunngenicity the subunit vaccines of Coxsackievirus (VP1), and to establish foundation for studying CA16 vaccine. Methods CA16 VP1 was amplified by RT-PCR and cloned into pFastBac HT A plasmid, recombinated with Bacmid DNA by transposition reaction and then transfected Sf9 cell, mixed with adjuvant AI(OH)_3. After immunization BALB/c mice, evaluating immune effectiveness after booster injections 2 weeks. Results The expressed protein was analyzed by SDS-PAGE and Western blot, mice immunized with CA16 (VP1) both induced specific IgG antibody and neutralization antibody. The best immunization antigen was 20 μg, IgG antibody was 1: 1600, neutralization antibody was 1:250, typical Th1/Th2 immune response was determined by lymphocyte proliferation assay and cytokine analysis. Conclusion The CA16 VP1 gene was cloned successfully and expressed in Sf9 insect cells, CA16 VP1 protein vaccine induced both humoral and cellular immune response, to lay solid foundation for further study on CA16 vaccine.

Objective To clone and express recombinant outer membrane protein P6, determine its optimal expression conditions and to investigate the immunoprotective effects of the P6 protein on mice. Methods The P6 gene of nontypeable Haemophilus influertzae(NTHi) was amplified by PCR from the NTHi genome and cloned into expression vector pET-32a (+) to generate the pET-32a-P6 recombinants. They were confirmed by nuclease digestion and sequence analysis. The verified recombinant was transformed into E. coli BL21 (DE3). Its optimal expression conditions were determined such as engineering strains, the concentration of IPTG, inducing temperature, inducing time, different medium etc. The recombinant protein was purified by Q Sepharose~(TM) XL ion exchange and gel filtration chromatography. The protein was analyzed by SDS-PAGE, Western blot and sequencing. BALB/c mice were immunized with recombinant protein be-fore challenged by NTHi through intraperitoneal injection. Then the mortality rate of different group was com-pared. Results The recombinant P6 of NTHi was successfully constructed and expressed in E. coli at a rel-atively high level. The purity was up to 95% after purification. The relative molecular mass of the protein is 14 145. 848. The recombinant protein was confirmed to show specific reaction on the antiserum through Western blot. The animal experiments showed the mortality rates of immunization groups were significantly lower than that of the control group (P < 0.05). Conclusion The successful expression of the recombinant P6 will be very helpful for the further study on development of vaccine, its purified, immunological activity and antibody preparation.

Objective To identify the variation in the Env region of SHIV-XJ02170 during passaging in Chinese origin Rhesus Macaques.Methods Fragments of the SHIV-XJ02170 gp160 and gp120 gene were amplified by PCR and RT-PCR separately from the blood samples of SHIV-XJ02170 infected animals at the peak viral load time point.Purified RT-PCR product was ligated into T easy vector and transformed into JM109 competent cells,18 clones were selected by PCR method and sequenced by ABI 3730DNA sequencers.The gene distances(divergence,diversity)were calculated using DISTANCE.Results In all,the SHTV-XJ02170 gp120 gene evolved forward along the virus passaging.It could be found that viral divergence from the founder strain serially enhanced during in vivo passaging,but in the early phase of each passage,SHIV-XJ02170 gp120 gene evolved toward ancestral state upon transmission to a new host.All of the SHIV-XJ02170 strains had V3 loop central motif(GPGQ)and were predicted to be using CCR5 on the basis of the critical amino acids within V3 loop.Conclusion There was significant increase in the genetic distance during serial passaging,and SHIV-XJ02170 gp120 gene evolved forward along passaging.This could partly explain why the virus infectivity was enhanced during in vivo passaging.

Objective To clone, express and purify the extracellular carbohydrate recognition do-main(CRD) of Dectin-1 in mouse peritoneal macrophages and to further investigate its ability to recognize and bind to β-glucans. Methods The Dectin-1 CRD gone was amplified by RT-PCR from RNA of mouse peritoneal macrophages and cloned into prokaryotic expression vector pET28a (+), the constructed pET-CRD recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and the fusion protein was in-duced to express. After affinity purification and renaturation, the fusion protein was incubated with Candida albicans yeast and its ability to recognize and bind to β-glucans in the cell wall of fungi. Results The fu-sion protein could recognize β-glucans in the fungal cell wall. Conclusion The recombinant expression plasmid pET28a-CRD was successfully constructed and the fusion protein was induced. The fusion protein is able to recognize and bind to β-glucans in the fungal cell wall, thus laying a good foundation for fungal de-tection and the exploration of the biological role of β-glucans.

Objective To establish the Flow-FISH method for simultaneous detection of telornere length and cell differentiation antigen. Methods HL60, Raji, Molt4 cells were cultivated. Each step and the conditions of the Flow-FISH procedure were optimized, standardized and validated, then 14 acute leukemia patients were observed for the changes of telomere length combined with differentiation antigen after complete remission by the method. Results Cells were stained with Alexa Fluor(R) 647-labeled antibody. Anti-gen-anfibedy complexes were covalenfly cross-linked onto the cell membrane before telomere staining. Cells were hybridized with telomere-specific fluorescein isothiocyanate (FITC)-conjugated peptide nucleic acid (PNA) probes followed by being counterstained with propidium iodide(PI). Multicolor Flow-FISH was performed to analyze telomere length and differentiation antigen simultaneously. The patients showed longer telomere and lower antigen expression after complete remission. Conclusion The Flow-FISH method for simultaneous detection of telomere length and differentiation antigen was successfully established which might prove to be a promising means for leukemia research especially in those without special molecular markers.

Objective To study the effects of purified rabies vaccine for human use (RV) on specific Th1/Th2 cytokines in human. Methods Twenty cases were injected intramuscularly with 5 full doses of RV. PBMCs were isolated from the blood sample collected at day 0, 14, 45 after the RV inoculation. Neutralizing antibody was determined by ELISA, and the proliferation of lymphocyte by in vitro test. The levels of RV specific IFN-γ, TNF, IL-2,IL-4, IL-5, IL-10 in the culture supernatants were detected by cytometric bead array (CBA). Results The neutralizing antibody was tested positive in 19 cases 45 days after inoculation and 1 case after 60 days, with the positive rate reaching 100%. After stimulation with RV, the lymphocyte transformation index at day 14, 45 in cases were significantly higher than those day of 0 (P< 0.05), and similar results were confirmed with IFN-γ, IL-2, IL-4, IL-5 tested by CBA (P<0.05). Condusion The RV could induce humoral and antigen-specific cellular immune responses in human, tested by showing good protective effect on rabies virus.

Objective To investigate the role of interleukin-17 (IL-17) in glomerulosclerosis secondary to adriamycin-induced nephropathy in rats. Methods Male Sprague-Dawley rats were divided into normal control group and adriarnycin-induced nephropathy group(model group). Eight rats were sacrificed at 2nd week, 4th week, 8th week, 12th week respectively. Histopathological changes of renal tissue were observed under light microscope and glomerulosclerosis index was evaluated. The expression of IL-17 was measured by RT-PCR, ELISA and immunohistochemistry. The expression of IL-1β was detected by ELISA. Results Compared with control group, the expressions of IL-17 mRNA, IL-17 and IL-1β protein were higher in model group of the 8th week (P=0.041, P=0.000 and P=0.007 respectively), even they were further higher in model group of the 12th week than that in the control group (all P=0.000). In model group of the 12th week, the level of IL-17 protein had positive correlation with IL-1β and glomerulosclerosis index (P=0.037, P=0.021 respectively). Conclusion IL-17 expression is elevated and may contribute to glomerulosclerosis in adriamycin-induced nephropathy.