OBJECTIVETo construct a prokaryotic expression system to serially express Alloferon-1 and to determine the anti-tumor activity of its products in vitro.

METHODSAn 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.

RESULTThe 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).

CONCLUSIONA 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.

Animals ; Anti-Bacterial Agents ; pharmacology ; Antineoplastic Agents ; metabolism ; pharmacology ; Bacterial Vaccines ; administration & dosage ; genetics ; metabolism ; Cell Line, Tumor ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Growth Inhibitors ; genetics ; metabolism ; pharmacology ; HL-60 Cells ; Helicobacter pylori ; genetics ; metabolism ; Humans ; KB Cells ; Peptides ; genetics ; immunology ; metabolism ; pharmacology ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacology

OBJECTIVETo 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.

METHODStpn17 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.

RESULTSThe 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%).

CONCLUSIONrTpN17-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.

Bacterial Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Genes, Bacterial ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Sensitivity and Specificity ; Syphilis ; diagnosis ; Syphilis Serodiagnosis ; methods ; Treponema pallidum ; genetics

OBJECTIVETo 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.

METHODSpIA-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.

RESULTS100% 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).

CONCLUSIONIn 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.

Antigens, Bacterial ; immunology ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; metabolism ; Base Sequence ; Enzyme-Linked Immunosorbent Assay ; methods ; Gene Expression Regulation, Bacterial ; Humans ; Neisseria gonorrhoeae ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

The computer-assisted surgery system is a complex system. All of the errors can be attributed to the loss of correspondence between the world coordinate system in the operation room and the virtual world coordinate system obtained from the multi-model medical images. The system's accuracy is composed of the accuracy of the localizer and that of registration. In order to improve the system accuracy, we analyse most of the possible error sources. The accuracy of the localizer affects deeply the registration between the intra-operation and pre-operation data. The localizer is the most basic and important part for a computer-assisted surgery system. We give a comprehensive possible error source at the end of the paper.
Algorithms ; Equipment Design ; Equipment Failure Analysis ; Imaging, Three-Dimensional ; instrumentation ; methods ; Magnetic Resonance Imaging ; Phantoms, Imaging ; Software ; Surgery, Computer-Assisted ; instrumentation ; methods ; Tomography, Emission-Computed, Single-Photon ; Tomography, X-Ray Computed

OBJECTIVETo 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.

METHODSUsing 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.

RESULTSIn 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.

CONCLUSIONA 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.

Animals ; Biopsy ; Capsid Proteins ; genetics ; Condylomata Acuminata ; pathology ; virology ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Human papillomavirus 11 ; genetics ; isolation & purification ; Human papillomavirus 6 ; genetics ; isolation & purification ; Humans ; Papillomavirus Infections ; virology ; Polymerase Chain Reaction ; Rabbits ; Sequence Homology, Nucleic Acid

Objective To investigate esophageal motility characteristics in gastroesophageal reflux disease (GERD) patients with or without dysphagia by high-resolution manometry and 24 h esophageal pH monitoring.Methods From August 2012 to November 2015,GERD patients with symptoms of acid reflux and heart burn who received 24 h esophageal pH monitoring were collected.The differences in esophageal motility were further analyzed between the GERD patients with dysphagia and without dysphagia.Student's t test,x2 test and Fisher's exact test were performed for comparison analysis.Results A total of 194 patients received 24 h esophageal pH monitoring and diagnosed as GERD,and at the same period completed esophageal high-resolution manometry.Among them,there were 17 GERD patients (8.8％) with dysphagia and 177 patients (91.2％) without dysphagia.The main classification of esophageal motility disorder of GERD patients with dysphagia was severe esophageal motility disorders (5/ 17),but the motility type of GERD patients without dysphagia patients mainly was mild esophageal motility disorders (10.2％,18/177).The integrated relaxation pressure,residual pressure of lower esophageal sphincter (LES),and contraction range at 3 cm and 11 cm above LES of GERD patients with dysphagia were all higher than those of patients without dysphagia ((9.70±0.98) mmHg (1 mmHg=0.133 kPa) vs (7.02±0.30) mmHg,(12.75±1.35) mmHg vs (9.18±0.42) mmHg,(106.80± 11.97) mmHg vs (70.82±3.48) mmHg,(82.66±10.70) mmHg vs (56.93±3.11) mmHg),and the differences were statistically significant (t=2.601,2.488,2.887,2.308,all P＜0.05).Distal esophageal contraction integral score of GERD patients with dysphagia was significantly higher than that of GERD patients without dysphagia ((2 128.94±310.47) mmHg · cm · s vs (1 029.88±90.16) mmHg · cm · s),and the difference was statistically significant (t =3.400,P =0.001).However,residual pressure of upper esophageal sphincter was significantly lower than that of patients without dysphagia ((2.84±1.21) mmHg vs (6.18±0.38) mmHg,t=-2.650,P=0.009).Conclusions Esophageal motility disorder of GERD patients with dysphagia is severer than that of patients without dysphagia.High resolution esophageal manometry can provide objective evidence of esophageal dynamics of GERD patients,which can guide the diagnosis and treatment of GERD.

Objective To construct a fusion gene (h1a-spaO) encoding H1a-SpaO protein of Sal-monella paratyphi A ( S.paratyphi A) and to express it in prokaryotic expression system , then to further ana-lyze the immunoprotective effects of the expressed protein rH 1a-SpaO.Methods The h1a-spaO fusion gene formed from separate h1a and spaO genes was amplified by PCR using flexible peptide sequence-containing linking primers and then sequenced after T-A cloning.A prokaryotic expression system for expressing h1a-spaO fusion gene was constructed by using the genetic engineering technique .The expressed protein rH1a-SpaO was examined by SDS-PAGE.The antigenicity and immunoreactivity of rH1a-SpaO protein were deter-mined by Western blot assay .The ability of rH1a-SpaO antiserum agglutinating S.paratyphi A strains was detected by micro-Widal′s test.The immunoprotective effects of rH 1a-SpaO against the lethal dose challenge of S.paratyphi A strains were analyzed in a mouse model and that were compared with those by using equal dose of individual recombinant protein H1a and SpaO (rH1a and rSpaO) as the immunogens, respectively. Results The h1a-spaO fusion gene was 100%identical with the individual h1a or spaO gene in nucleotide and amino acid sequences .The constructed prokaryotic expression system could express the recombinant pro-tein rH1a-SpaO with an advantage of high efficiency .rH1a-SpaO protein was able to react with rH 1a or rSpaO antiserum.Moreover, rH1a-SpaO antiserum also could efficiently recognize rH 1a and rSpaO as well as agglutinate Salmonella paratyphi A strains by binding with H-antigen.The immunoprotective rate (93.3%) in mice pre-immunized with 100 μg of rH1a-SpaO protein was significantly higher than that in those pre-immunized with equal dose of rH1a (60.0%) protein or rSpaO protein(53.3%) (P<0.05).Conclusion The recombinant fusion protein rH 1a-SpaO showed more stronger immunoprotective function than the individ-ual rH1a or rSpaO protein , which could be used as an effective antigen for the development of bi -valent para-typhoid A vaccine or typhoid/paratyphoid capsular polysaccharide-protein combined vaccine .

Objective To observe the dynamic changes of neuron-specific enolase(NSE)mRNA and protein in offspring rats brain tissue with bilirubin encephalopathy and explore the pathological mechanism and its diagnostic value on bilirubin encephalopathy.Methods Seven-day postnatal Wistar rats were used for study.One hundred and twenty rats were divided into 2 groups randomly(control group and experimental group),which were respectively subdivided into 6 groups(6,12,24,48,72,96 h).The rats in control group were intraperitoneally administered physiological saline 0.5 mL,the rats in experimental groups were intraperitoneally administered bilirubin(200 mg/kg).Reverse transcription-polymerase chain reaction was used to analyze the dynamic changes of NSE mRNA expression at 6,12,24,48,72 and 96 h in brain tissue of rats with bilirubin encephalopathy.Immunohistochemistry was used to evaluate NSE protein expression in hippo-campi,cerebral cortex,thalamic and pallidus at different times.Results The expression of NSE mRNA significantly decreased in brain tissue of rats with bilirubin encephalopathy from 6 h to 96 h compared with the control groups.The expression of NSE protein in hippocampi decreased in offspring rats with bilirubin encephalopathy from 6 h to 96 h,but there were no differences compared with the control groups.The expression of NSE protein in cerebral cortex was significantly decreased in rats with bilirubin encephalopathy from 6 h to 96 h,there were significant differences compared with the control group.The expression of NSE protein in thalamic significantly decreased in rats with bilirubin encephalopathy from 6 h to 96 h,but there were significant differences between experimental groups and the control groups at 24 h and 72 h.The expression of NSE protein in pallidus significantly decreased in offspring rats with bilirubin encephalopathy from 6 h to 96 h,and there were significant differences compared with control groups.Conclusions The changing trends of expression of NSE mRNA were identical to those of NSE protein.NSE may reflect the degree of injury of neurogliocyte.It can serve as reliable index to determine bilirubin encephalopathy.

OBJECTIVETo determine the adhering ability of Leptospira interrogans to extracellular matrix molecules (ECM) of host cells and its diversity.

METHODSThe 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.

RESULTL. 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.

CONCLUSIONL. interrogans adheres to host cells through ECM molecules; LN, FN, DEN, COL1, COL2 and COL4 are the receptor molecules with different adhesion intensity.

Animals ; Bacterial Adhesion ; Cercopithecus aethiops ; Decorin ; Extracellular Matrix ; metabolism ; microbiology ; Extracellular Matrix Proteins ; metabolism ; Fibronectins ; metabolism ; Laminin ; metabolism ; Leptospira interrogans ; pathogenicity ; Macrophages ; microbiology ; pathology ; Mice ; Proteoglycans ; metabolism ; Vero Cells

OBJECTIVETo investigate the pathogenicity of Leptospira interrogans fliR gene to J774A.1 cells.

METHODSfliR 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.

RESULTThe 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).

CONCLUSIONfliR 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.

Animals ; Apoptosis ; Bacterial Adhesion ; Bacterial Proteins ; genetics ; metabolism ; Cell Line ; Leptospira interrogans ; genetics ; pathogenicity ; Macrophages ; microbiology ; pathology ; Membrane Proteins ; genetics ; metabolism ; Mice ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics