Molecular Characterization and Correlation with β-lactam Resistance of Streptococcus pneumonia Isolates in Hangzhou, China / 生物医学与环境科学（英文）

Penicillin-binding proteins (PBPs) are the target of β-lactam antibiotics (the major treatment for Streptococcus pneumoniae infections), and mutations in PBPs are considered as a primary mechanism for the development of β-lactam resistance in S. pneumoniae. This study was conducted to investigate the mutations in the PBPs of clinical S. pneumoniae isolates in Hangzhou, China, in correlation with β-lactam resistance. Results showed that 19F was the predominant serotype (7/27) and 14 of the S. pneumoniae isolates were resistant to both penicillin G and cephalosporin. Genotyping results suggested that β-lactam-resistant isolates primarily exhibited single-site mutations in both the STMK and SRNVP motifs of pbp1a in combination with double-site mutations in the STMK motif of pbp2x, which might be the primary mechanisms underlying the β-lactam resistance of the isolates in this study.

Distribution of Salmonella paratyphi A outer membrane protein X gene and immune-protective effect related to its recombinant expressed products / 中华流行病学杂志

Objective To determine the distribution and sequence conservation of outer membrane protein X (ompX) gene in Salmonella paratyphi A isolates as well as the immunogenicity and irnmono-protection of ompX gene products.Methods OmpX gene in Salmonella paratyphi A isolates was detected by PCR and the amplification products were sequenced after the T-A cloning process.OmpX gene product was expressed with E.coli expression system and the expressed rOmpX was extracted by Ni-NTA affinity chromatography.SDS-PAGE and Bio-Rad Gel Image Analyzer were applied to examine the expression and yield of rOmpX.Both antigenicity and immune-reactivity of rOmpX were detected by immune-diffusion test,ELISA and Western blot assay.The immuneprotective effect of rOmpX against infection of Salmonella paratyphi in mice was determined and the agglutinative titers of sera from rOmpX-immunized mice was measured by micro-Widal' s test.Results All the tested Salmonella paratyphi A isolates had ompX gene with high nucleotide or amino acid sequence identity (99.2％-100.0％ or 98.4％-100.0％).When rOmpX was induced to rabbits to produce high level antibody and combined with antiserum against whole cell of Salmonella paratyphi A,the results displayed a positive Western hybridization signal.Results from ELISA demonstrated that 95.6％ (65/68) of the serum samples from paratyphoid-A patients were positive on rOmpX antibody.Mice that were immunized with 100 μg or 200 μg rOmpX displayed an immune-protective rate of 93.3％ (14/15) or 100.0％ (15/15).Sera from those rOmpX-immunized mice provided 1 ∶ 10-1 ∶ 40 agglutination titers in both H antigens of Salmonella paratyphi A and Salmonella typhi.Conclusion The recombinant expression product of ompX gene could be used as a candidate antigen for developing genetic engineering vaccines against Salmonella paratyphi A infection.

Detected of associated contagious parameters of blood recipients before transfusion and their clinical significance / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the significance in prevention of nosocomial infection of the testing of the associated contagious parameters of blood recipients before transfusion.</p><p><b>METHODS</b>A retrospective analysis was adopted, 44 968 pre-transfusion patients were tested the serum hepatitis B virus surface antigen (HBsAg), antibody against hepatitis C virus (anti-HCV), antibody against T. pallidum (anti-TP) and antibody against human immunodeficiency virus(anti-HIV).</p><p><b>RESULTS</b>The total positive rate was 22.41%. Positive rate of HBsAg, anti-HCV and anti-TP were 20. 67% (9294/44 968) , 0.33% (148/ 44 968) and 1.65% (9741/44968), respectively; anti-HIV was positive in 39 patients, 23 cases coinfection of the other three indicators at least one positive in 39 cases of anti-HIV-positive blood recipients, of which was mostly observed T. pallidum; co-infection of HBV, HCV and/or TP were 117 cases, and were mostly observed between HBV and HCV, HCV and TP; for HBV infection the department of digestive medicine was prevalent(Chi2>or=83.0, P <0.01).</p><p><b>CONCLUSION</b>Part of blood recipients before admission had been infected with a contagious disease. The testing of the associated contagious parameters of blood recipients before transfusion is not only useful for both of the hospital and the patients, but also more important to ensure safe blood transfusion, decrease medial dissatisfaction and to prevent nosocomial infection.</p>

Evaluation of specific antibodies of blood receiver sera to Treponema pallidum by chemiluminescence immunoassay / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The purpose of this study was to evaluate the diagnostic performance of chemiluminescence immunoassay (CLIA ) , in comparison with that of the following currently used treponemal tests: hemagglutination test (TPHA), and Western Blot (WB).</p><p><b>METHODS</b>First, a retrospective study was performed with a panel of 18 494 blood receiver sera by CLIA and TPHA, the specific antibody against T. pallidum in 177 were positive sera by CLIA and/or TPHA, 81 clinical and serologically characterized syphilitic sera, 55 sera obtained from subjects with potentially interfering diseases, and 250 healthy sera were negative were detected by CLIA, TPHA and WB.</p><p><b>RESULTS</b>The results of WB as the gold standard, the sensitivity of CLIA (98. 4%) was significantly higher than that of TPHA (94. 4%) (Chi2 = 5.76,P <0. 05), the specificity of CLIA (100%) was similar to that of TPHA (99.7%) (Chi2 =1. 0, P >0. 05).</p><p><b>CONCLUSION</b>CLIA is characterized with higher sensitivity and specificity, It is suitable for screening Syphilis in clinical laboratory.</p>

CagA(+) H. pylori induces Akt1 phosphorylation and inhibits transcription of p21(WAF1/CIP1) and p27(KIP1) via PI3K/Akt1 pathway / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Cytotoxin-associated protein (CagA) of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we try to determine roles of CagA(+) strain in activating PI3K/Akt1 signaling pathway, and affecting expression of p21(WAF1/CIP1) and p27(KIP1), and also in releasing IL-8 in host cells.</p><p><b>METHODS</b>Akt1 phosphorylation and IL-8 levels of CagA(+) and CagA⁻ strain infected AGS cells were detected by ELISAs. Two quantitative RT-PCRs were established to measure p21(WAF1/CIP1) and p27(KIP1) mRNA levels in the CagA(+) and CagA⁻ strain infected cells. LY294002, an inhibitor of PI3K/Akt pathway, was used to define effect of the pathway in IL-8 release.</p><p><b>RESULTS</b>CagA(+) strain could induce an obvious elevation of Akt1 phosphorylation in the infected AGS cells while CagA? strain failed to do so. The CagA(+) H. pylori strain infected AGS cells showed significant drops both in p21(WAF1/CIP1) and p27(KIP1) mRNA levels, whereas the CagA⁻ H. pylori strain caused a remarkable increase in p21(WAF1/CIP1) mRNA without affecting p27(KIP1) gene transcription in the AGS cells. Both the CagA(+) and CagA⁻ H. pylori strains enabled AGS cells to produce close elevated levels of IL-8, and the LY294002 block resulted in unexpected elevations of IL-8 levels.</p><p><b>CONCLUSIONS</b>CagA can activate PI3K/Akt1 pathway that plays an inhibitory role in IL-8 release in H. pylori infected AGS cells. Activation of PI3K/Akt1 pathway and subsequent negative regulation of p21(WAF1/CIP1) and p27(KIP1) expression might be involved in CagA-associated carcinogenesis.</p>

Rapid detection of rpoB gene mutations in rif-resistant M. tuberculosis isolates by oligonucleotide microarray / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To detect the specific mutations in rpoB gene of Mycobacterium tuberculosis by oligonucleotide microarray.</p><p><b>METHODS</b>Four wild-type and 8 mutant probes were used to detect rifampin resistant strains. Target DNA of M. tuberculosis was amplified by PCR, hybridized and scanned. Direct sequencing was performed to verify the results of oligonucleotide microarray.</p><p><b>RESULTS</b>Of the 102 rifampin-resistant strains 98 (96.1%) had mutations in the rpoB genes.</p><p><b>CONCLUSION</b>Oligonucleotide microarray with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of rifampin resistance in M. tuberculosis isolates.</p>

Recombinant expression of Streptococcus pneumoniae comD/E/C genes and correlation of ComD/C with beta-lactam antibiotic resistance / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct prokaryotic expression systems of TCS genes comD/comE/comC of Streptococcus pneumoniae, and to determine the correlation of ComD and ComC with the drug resistance.</p><p><b>METHODS</b>The entire comD, comE and comC genes were amplified by PCR and their prokaryotic expression systems were established by routine genetic engineering technique. SDS-PAGE and Bio-Rad Agarose Image Analyzor was applied to measure the outputs of target recombinant proteins rComD, rComE and rComC. Rabbits were immunized with these recombinant proteins to prepare antisera. The resistance of S.pneumoniae strains to penicillin and cefotaxime was examined after ComD and ComC were blocked by antisera.</p><p><b>RESULT</b>Compared with the reported sequences, similarities of nucleotide and amino acid sequences of the cloned comD, comE and comC genes were 98.4% approximately 99.3% and 99.1% approximately 100%, respectively. The constructed engineering bacteria E.coli BL21DE3(pET42a-comD), E.coli BL21DE3(pET42a-comE) and E.coli BL21DE3(pET42a-comC) were able to efficiently express the target recombinant proteins and the outputs of rComD, rComE and rComC were 28%, 25% and 35% of the total bacterial proteins, respectively. The double immunodiffusion titers of rabbit antisera against rComD, rComE or rComC were 1:4, 1:4 and 1:8, respectively. After the ComD and/or ComC were blocked by the antisera, the cefotaxime-sensitive S. pneumoniae strains became to resistant to antibiotics but there were no changes for cefotaxime-resistant strains and resistance to penicillin for all tested strains.</p><p><b>CONCLUSION</b>The prokaryotic expression systems of S.pneumoniae comD/come/comC genes have been successfully constructed, and the study also indicates that both the ComD and ComC are involved in the drug resistance of S. pneumoniae to cefotaxime.</p>

Serial recombinant expression and activity against tumor cells in vitro of antibacterial peptide Alloferon-1 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct a prokaryotic expression system to serially express Alloferon-1 and to determine the anti-tumor activity of its products in vitro.</p><p><b>METHODS</b>An artificial fusion gene containing 6 x His-EK-8 x Alloferon-1-EK-6 x His sequences was constructed by linking primer PCR. By using routine molecular biological methods, the artificial fusion gene was cloned and its prokaryotic expression system was then constructed. SDS-PAGE and BioRad Agarose Image Analysor was applied to measure the expression and output of the target recombinant products 8 x rAlloferon-1-EK. Ni-NTA affinity chromatography and EK digestion and Sephadex G-50 chromatography were performed to extract 8 x rAlloferon-1-EK and rAlloferon-1-EK, respectively. The proliferation of KB, SGC and HL-60 tumor cells was tested by using MTT method after treatment with directly synthesized Alloferon-1 (sAlloferon-1), Aloferon-1-EK (sAlloferon-1-EK) and rAlloferon-1-EK.</p><p><b>RESULT</b>The target artificial fusion gene and its prokaryotic expression system pET42a-8 x rAlloferon-1-EK-E. coliBLDE3 with the expected sequences were obtained. Under inducement of IPTG, the prokaryotic expression system expressed the target serial recombinant protein 8 x rAlloferon-1-EK and its output was approximate 30 % of the total bacterial proteins. 8 x rAlloferon-1-EK and rAlloferon-1-EK were obtained through Ni-NTA and Sephadex G-50 columns. sAlloferon-1, sAlloferon-1-EK and rAlloferon-1ìrAlloferon-EK showed similar remarkable effects of inhibiting the growth and proliferation of KB, SGC and HL-60 cells in vitro within 25 approximately 100 microg/ml concentration range (P<0.01), and there were no significant differences in the inhibiting effects among the three agents (P>0.05).</p><p><b>CONCLUSION</b>A prokaryotic expression system to serially express rAlloferon-1 has been successfully constructed. The product rAlloferon-1-EK has a similar anti-tumor activity compared to both the synthesized Alloferon-1 and Alloferon-1-EK in vitro.</p>

Comparison of serological detection effects of ELISA using rTpN17 or rTpN47 of Treponema pallidum as antigen with that of TPHA and TRUST / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To clone tpn17 and tpn47 genes of Treponema pallidum and then construct their prokaryotic expression systems,to establish ELISAs based on rTpN17 and rTpN47 as antigens and to evaluate the sensitivity and specificity of the ELISAs for detection of serological diagnosis of syphilis.</p><p><b>METHODS</b>The whole length of tpn17 and tpn47 genes was amplified by PCR and then their prokaryotic expression systems were constructed. SDS-PAGE was used to measure the expression of the target recombinant proteins rTpN17 and rTpN47. Ni-NTA affinity chromatography was applied to extract rTpN17 and rTpN47, while Western blot was performed to determine the specific immunoreactivity of rTpN17 and rTpN47. By using rTpN17 and rTpN47 as the coated antigen, respectively, ELISAs (rTpN17-ELISA and rTpN47-ELISA) were established to detect serum samples from 200 healthy individuals, 25 RA patients, 17 SLE patients and 211 syphilis patients. The detection effects of the ELISAs were compared to those of TRUST and TPHA.</p><p><b>RESULT</b>The sequence similarity of the cloned tpn17 and tpn47 genes was 100 % compared with the corresponding sequences in GenBank. The expression outputs of rTpN17 and rTpN47 were approximately 37.2 % and 26.8 % of the total bacterial proteins, respectively. Both the extracted rTpN17 and rTpN47 could take place remarkable conjugation reactions to the sera with positive antibody against Treponema pallidum.The positive detection rate of TPHA (99.1%) was the highest (P<0.001). The positive detection rates of rTpN17-ELISA (85.3 %) and rTpN47-ELISA (84.3 %) were similar (P>0.05). The positive detection rates of TRUST (72.5 %) was lower than that of rTpN17-ELISA (P=0.001) but similar to that of rTpN47-ELISA (P=0.014). The detection results of all the serum samples from healthy individuals, RA patients and SLE patients were negative, whereas 7.1 % (3/42) of the samples from RA or SLE patients were positive.</p><p><b>CONCLUSION</b>rTpN17 and rTpN47 are still maintaining their original immunoreactivity. The ELISAs using rTpN17 or rTpN47 as the antigen are rapid, simple and convenient, higher sensitivity and specificity methods for serological screening and detection of syphilis.</p>

Establishment and application of enzyme linked immunosorbent assay based on the outer membrane pIA-pIB fusion gene of Neisseria gonorrhoeae / 中华流行病学杂志

<p><b>OBJECTIVE</b>To clone pIA and pIB genes of Neisseria gonorrhoeae,and to construct pIA-pIB fusion gene and its prokaryotic expression system, and to establish enzyme linked immunosorbent assay (ELISA) based on rPIA-PIB for detecting serum and pus samples from gonorrhea patients and to evaluate the sensitivity and specificity of the ELISA.</p><p><b>METHODS</b>pIA-pIB fusion gene was constructed by polymerase chain reaction (PCR) using linking primers and a prokaryotic expression system of the fusion gene was constructed by using routine molecular biological methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) plus BioRad Gel Image Analyzer was used to measure the expression of the target recombinant protein rPIA-PIB. Ni-NTA affinity chromatography was performed to extract and purify rPIA-PIB. An ELISA by using rPIA-PIB as the coated antigen for detecting the specific IgG against rPIA and/or rPIB in gonorrhea patients' sera as well as another ELISA by using rPIA-PIB antiserum as the first antibody for detecting the rPIA and/or rPIB in gonorrhea patients' pus samples were established. In these experiments, ELISAs associated with rPIA, rPIB and their antisera were applied as the controls.</p><p><b>RESULTS</b>100% similarities of the nucleotide and putative amino acid sequences of the pIA-pIB fusion gene were confirmed when compared with the original sequences. The output of rPIA-PIB was 29.8% of the total bacterial proteins. The purified rPIA-PIB only showed a single target protein segment in gel after SDS-PAGE. Using a positive rate (98.3%) of rPIA-PIB-IgG-ELISA to detect 119 cases of gonorrhea patients' serum samples was remarkably higher than that of rPIA-IgG-ELISA (30.3%) or rPIB-IgG-ELISA (66.4%) (P<0.01). The positive rate (91.6%) of rPIA-PIB-ELISA to detect 119 cases of gonorrhea patients' pus samples was also significantly higher than that of rPIA-IgG-ELISA (27.7%) or rPIB-IgG- ELISA (62.2%) (P<0.01).</p><p><b>CONCLUSION</b>In this study we successfully constructed pIA-pIB fusion gene of N. gonorrhoeae and its prokaryotic expression system while rPIA-PIB showed obvious superiority used as the antigen in gonorrhea associated detection kits compared to both the rPIA and rPIB.</p>

Association of fliR gene in Leptospira interrogans with adhesion and pathogenicity to host cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the pathogenicity of Leptospira interrogans fliR gene to J774A.1 cells.</p><p><b>METHODS</b>fliR gene from L. interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 and kana gene from plasmid pET42a were amplified by PCR. Suicide plasmid of fliR gene was constructed; and specific siRNA for fliR gene was designed and synthesized. fliR gene mutants were constructed by gene knock-out with suicide plasmid (56601fliR-Kana) and gene silencing with siRNA (56601siRNA-R2). The mutants were identified by PCR, sequencing and semi-quantitative RT-PCR. Adhesion to mouse mononuclear-macrophage J774A.1 and induction of cell necrosis and apoptosis by 56601fliR-Kana and 56601siRNA-R2 were examined by adhesion test and flow cytometry, respectively.</p><p><b>RESULT</b>The nucleotide and putative amino acid sequences of cloned fliR gene had 99.9% and 100% similarities to those of reported sequences in GenBank. The nucleotide sequence of the cloned kana gene was identical to the corresponding sequence in pET42a map. The results of PCR and sequencing confirmed that kana gene was inserted in the sequence of 56601fliR-Kana fliR gene. The mRNA level of fliR gene in 56601fliR-Kana was remarkably decreased (P<0.01) while the mRNA level of fliR gene in 56601siRNA-R2 was much lower than that in wild strain 56601 (P<0.05). 56601fliR-Kana and 56601siRNA-R2 lost the ability to adhere J774A.1 cells; and their ability to induce cell necrosis and apoptosis was markedly weakened (P<0.01).</p><p><b>CONCLUSION</b>fliR is a virulence-associated gene of L. interrogans and the function of the gene is closely related to adhesion, induction of cell necrosis and apoptosis of the microbe.</p>

Adhering effect of Leptospira interrongan to major extracellular matrix molecules / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine the adhering ability of Leptospira interrogans to extracellular matrix molecules (ECM) of host cells and its diversity.</p><p><b>METHODS</b>The infection models of L. interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 to J774A.1, L929 and Vero cells were established. Fontana silver impregnation method was applied to demonstrate the adhering effect of microbes to extracellular matrix (ECM) of the cells. ELISA methods were established to detect the adhering effects of L. interrogans to ECM molecules laminin (LN), fibronectin (FN), decorin (DEN) and collagen1, 2, 4 (COL1, 2, 4). A competitive inhibition test was performed to verify the results.</p><p><b>RESULT</b>L. interrogans strain 56601 adhered the ECMs of L929, J774A.1 and Vero cells with its one or two sites. L. interrogans strain 56601 adhered all six ECM molecules; the adhering effects to COL1, LN, COL4 were relatively stronger. The adhering effects were markedly decreased after the microbes were pre-incubated with corresponding ECM molecules.</p><p><b>CONCLUSION</b>L. interrogans adheres to host cells through ECM molecules; LN, FN, DEN, COL1, COL2 and COL4 are the receptor molecules with different adhesion intensity.</p>

Recombinant expression of Streptococcus pneumoniae ciaH/R genes and their correlation with beta-lactam antibiotic resistance / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct prokaryotic expression systems of Streptococcus pneumoniae ciaH and ciaR genes,and to determine their correlation with drug resistance.</p><p><b>METHODS</b>The total length of ciaH and ciaR genes was amplified by PCR and their prokaryotic expression systems were established by using routine genetic engineering technique. SDS-PAGE was applied to measure the outputs of target recombinant proteins rCiaH and rCiaR. Rabbits antisera and IgGs against rCiaH and rCiaR were prepared. The resistance to penicillin and cefotaxime of S.pneumoniae strains was examined after CiaH and CiaR were extracellularly and intracellularly blocked by the IgGs.</p><p><b>RESULT</b>The homogeneity of nucleotide and amino acid sequences of the cloned ciaH and ciaR genes with the reported sequences was 99.9-100% and 100%, respectively. The recombinant bacteria E.coli BL21DE3pET42a-ciaH and E.coli BL21DE3pET42a-ciaR were able to express the target recombinant proteins rCiaH and rCiaR with efficiency. The outputs of rCiaH and rCiaR were 33% and 45% of the total bacterial proteins, respectively. The double immunodiffusion titers of rCiaH antiserum,rCiaR antiserum,rCiaH-IgG and rCiaR-IgG were 1:4,1:4,1:1 and 1:1, respectively. After CiaH was extracellularly or intracellularly blocked by CiaH-IgG, and CiaR was intracellularly blocked by CiaR-IgG, the penicillin-sensitive or cefotaxime-sensitive strains developed resistance to the two antibiotics; but the blocks did not change that of penicillin-resisting or cefotaxime-resisting strains.</p><p><b>CONCLUSION</b>The prokaryotic expression systems of S. pneumoniae ciaH/ciaR genes have been successfully constructed in this study. Both the CiaH and CiaR may be involved in penicillin and cefotaxime resistance of the bacterium.</p>

Study on ELISAs with rTpN17, rTpN47 and its fusion protein as antigens in the detection of serum samples from syphilis patients / 中华流行病学杂志

<p><b>OBJECTIVE</b>To construct the prokaryotic expression systems of tpnl7 and tpn47 genes and tpn17-tpn47 fusion of Treponema pallidum, and to establish ELISAs based on rTpN17, rTpN47 and rTpN17-TpN47 as antigens to evaluate the sensitivity and specificity of the ELISAs for detection of serological diagnosed syphilis.</p><p><b>METHODS</b>tpn17 and tpn47 genes were amplified and cloned by routine molecular biological methods. PCR with linking primers was used to construct artificial fusion gene tpn17-tpn47. The prokaryotic expression systems of the genes were then constructed. SDS-PAGE was used to measure the expression of the target recombinant proteins rTpN17, rTpN47 and rTpN17-TpN47. Ni-NTA affinity chromatography was applied to extract the three recombinant proteins, while Western blot was performed to determine their immunity. Using rTpN17, rTpN47 and rTpN17-TpN47 as the coated antigens, ELISAs (rTpN17-ELISA, rTpN47-ELISA and rTpN17-TpN47- ELISA) were established to detect serum samples from 200 healthy individuals, 25 RA patients, 17 SLE patients and 419 syphilis patients. Results of the ELISAs were compared to those with TRUST and TPHA.</p><p><b>RESULTS</b>The sequence similarities of the cloned tpnl7 and tpn47 genes and the constructed tpn17-tpn47 fusion gene were 100%, compared with the corresponding sequences in GenBank. The expression outputs of rTpN17, rTpN47 and rTpN17-TpN47 were 37.2%, 23.3% and 29.8% of the total bacterial proteins, respectively. Each of the three purified recombinant proteins showed a single fragment in gel after electrophoresis, and could take place remarkable conjugation reactions to the positive sera from syphilis patients. The detection results of rTpN17-ELISA, rTpN47-ELISA and rTpN17-TpN47-ELISA were negative for the serum samples from healthy individuals, RA and SLE patients, while presented 84.4%, 82.3% and 98.1% positive detection rates for the serum samples from syphilis patients. The positive detection rates of rTpN17-ELISA and rTpN47-ELISA were lower than that of TPHA (P<0.01), while the positive detection rate of rTpN17-TpN47-ELISA was similar to that of TPHA (P>0.05). All the positive detection rates from ELISA tests were higher than that of TRUST (71.4%).</p><p><b>CONCLUSION</b>rTpN17-ELISA, rTpN47-ELISA and especially rTpN17-TpN47-ELISA established in this study were of great hope as it was rapid, simple, convenient, safe, with high sensitivity and specificity for serological screening and detection of syphilis.</p>

MRNA expression of complement C3 and C4 in rat nasal mucosa with allergic rhinitis / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the level of mRNA expression of complement C3 and C4 in rat nasal mucosa and to reveal the relationship with the pathogenesis of allergic rhinitis (AR) .</p><p><b>METHODS</b>Twenty healthy SD rats were randomly divided into AR group and control group, 10 rats for each group. Ten rats was sensitized and intranasally challenged by ovalbumin and Al (OH)3 (as supplement) as allergic rhinitis models, and the control group was treated by saline. RT-PCR was performed to investigate the level of mRNA expression of complement C3 and C4 in nasal mucosa of both groups.</p><p><b>RESULTS</b>C3 and C4 mRNA were detected in both groups. The relative intensity of gene expression was measured. The relative intensity of C3 mRNA expression was 6183+/-1376 in AR group, 4444+/-989 in control group, C4 mRNA was 4398 +/-948 in AR group, and 2771+/-407 in control group. Expression of C3 and C4 in AR group was higher than that of the controls ( P < 0. 05) .</p><p><b>CONCLUSION</b>The high level of C3 and C4 mRNA expression in nasal mucosa of rats with allergic rhinitis suggests that C3 and C4 are involved in the immunopathology of allergic rhinitis. The result implies that complement system involved in the rat's allergic rhinitis is possibly activated through the classical pathway.</p>

Study on the frequency of human papillomavirus type 6 and type 11 infection and L1 gene expression of the virus in biopsy samples of pointed condyloma patients / 中华流行病学杂志

<p><b>OBJECTIVE</b>To determine the different rates of human papillomavirus types 6 (HPV-6) and 11 (HPV-11) infection in biopsy samples from pointed condyloma patients, and to construct prokaryotic expression system of the major capsid protein L1 of the virus so as to establish an ELISA for detecting the expression of L1 gene in the biopsy samples.</p><p><b>METHODS</b>Using a double PCR based on the L1 gene of HPV-6 and HPV-11, the infection rates of HPV-6 and HPV-11 in the biopsy samples were determined. The whole length of HPV-6 L1 gene was amplified using PCR and the target amplification fragment was sequenced after T-A cloning. The prokaryotic expression system pET32a-L1-E. coli BL21 (DE3) was constructed and SDS-PAGE was used to measure the expression of the target recombinant protein rL1. Rabbit anti-rL1 serum was prepared and immuno-diffusion assay was applied to examine the antiserum titer. ELISA was established to detect the expression of L1 gene in the biopsy samples.</p><p><b>RESULTS</b>In the biopsy samples from 116 pointed condyloma patients, 92.2% (107/116) were detectable for HPV-6 and/or HPV-11. Of the 107 positive samples, 70.1% (75/107) and 23.4% (25/107) were positive for HPV-6 or HPV-11 alone and 6.5% (7/107) were coinfected with both HPV-6 and HPV-11 respectively. When compared with the reported corresponding sequences, the homology of nucleotide and sequence of the cloned HPV-6 L1 gene was from 99.20% - 99.93% while its putative amino acid sequence homology was from 99.80% - 100%, suggesting IPTG could induce the expression of rL1. The immuno-diffusion titer of the rabbit anti-rL1 serum was 1:4. 88.8% (103/116) of the biopsy samples were the major capsid protein L1 detectable.</p><p><b>CONCLUSION</b>A prokaryotic expression system of HPV-6 L1 gene, a double PCR assay for HPV-6 and HPV-11 genotyping, and an ELISA assay for detecting the major capsid protein L1 were successfully established in this study. The pointed condyloma patients in Zhejiang area mainly infected with HPV-6. The HPV in the focus frequently expressed major capsid protein L1.</p>