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Objective To investigate the prevalence and resistance mechanisms of Proteus mirabilis in the ward of neurology de‐partment of our hospital .Methods For a total of 20 clinic isolates of Proteus mirabilis ,PCR were used for the detection of AmpC , ESBLs ,KPC and MBLs and then DNA sequencing was performed .The integrons were also detected by using PCR and then sequen‐cing was carried out .The genetic relationship between isolates were detected and analysed by pulsed‐field gel electrophoresis(PF‐GE) .The results of drug sensitivity tests were analysed .Results TEM‐1 and CTX‐M‐14 gene were found in all the 20 isolates ,the 10 isolates of Proteus mirabilis were also found carrying CMY‐2 gene .Class Ⅰ integrons were amplified from 19 strains carrying gene cassettes aacA4+cmlA1,dfrA12+orfF+aadA2and dfrA32+ereA+aadA2 respectively .PFGE analysis revealed that the 20 isolates were grouped into 11 PFGE types P1-P11 ,the 12 isolates of P1-P3 were same clones .The sensitive rates of the i‐solates to Meropenem ,Amikacin ,Aztreonam ,Ceftazidime and Tazocin were high .Conclusion Nosocomial transmission of the same clone of Proteus mirabilis was appeared in the ward of neurology department of our hospital .The predominance drug‐resistance genes were CTX‐M‐14 andCMY‐2 .The incidence of carrying class Ⅰ integrons was high ,and the major gene cassettes wereaacA4+cmlA1and dfrA12+orfF+aadA2.The 20 isolates were all sensitive to Meropenem ,Amikacin and Aztreonam .Other Clinical departments should also pay attention to the nosocomial infection caused by Proteus mirabilis and strengthen the infection control measures .
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Objective To preliminarily evaluate the performance of glucose detention reagents with three kinds of different chro-mogens and to investigate their anti-interference performance according to NCCLS document.Methods According to the protocol EP10-A2 provided by the National Committee for Clinical Laboratory Standards(NCCLS),the samples with low,middle and high level of glucose were detected by the glucose reagent kits with 3 kinds of different chromogens.The bias,total imprecision,inter-cepts,slope rates,nonlinearities,carryover rate and drifts were calculated.The interference evaluation test was performed according to the document EP7-A2.Results The bias and total imprecision of three kinds of reagent kits were all within allowed ranges.No statistically significant differences were showed in intercepts,slope rates,nonlinearities,carryover rate and drifts.1450 turbidity chyle,5 g/L hematoglobin and 0.03 g/L vitamin C did not interfere with the assay of three kinds of glucose reagent kits with differ-ent chromogens.342 μmol/L free bilirubin,342 μmol/L conjugated bilirubin did not interfere with the detection of reagent with MAOS.Conclusion The glucose detention reagents with three different chromogens have good accuracy and precision,and various performance indexes all conform to the clinical application requirements,reagent with chromogen MAOS is better than other chro-mogenic reagents in the anti-interference performance.
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Objective To clone and express human autoantigen Sm B'in methylotrophie yeast Pichia Pagtoris.Methods The gene Sm B' was cloned bv PCR The PCR product wag inserted into the vector pPIC9k.The recombinant plasmid pPIC9k.Sm B' was transformed into yeast Sm D1168 by electroporation.The positive clones were screened in MD plates.The high copy number transformants were rapidly selected by using G418 and were induced by methan01.Supematants after induction were analyzed by SDS-PAGE and western blot.Sera collected from thirty patients with SLE.thirty patients with mixed connective tissue disease(MCTD)and thirty healthy volunteers were detected by immunodot and immunoblot.Results The PCR product wag about 700 bD in size which Wag in accordance with predicted 657 bp.The pPIC9k-Sm B'showed the same seqencing result with GenBank's report and restriction enzyme analysis confirmed our prediction.The pPIC9k-Sm B' positive clone produced a 32 000 protein which had natural immunogenicitv of human autoantigen Sm B'by SDS-PAGE and western blot.The positive rate of immunodot and IBT were 46.7%(42/90)and 51.1%(46/90),respectively.The agreement between immunodot and IBT was very close(Kappa value=0.911 2,P＜0.01).Conclusion Successfully cloning and expression of human autoantigen Sm B' in methylotmphic yeast Pichia Pagtoris hid a foundation for further research work.
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To obtain the aptamers with high affinity and specificity for cyclosporin A(CsA),a synthesized 78 nt single stranded DNA(ssDNA)random library containing 35 random sequences flanked by invariant primer was subjected to 1 1 rounds of selection against CsA by SELEX protocol.Magnetic beads were used for target immobilization and the biotin-streptavidin-horseradish peroxidase system was employed for determining the binding affinity between the aptamers and CsA. After ten rounds of selection and amplification, with an increasing affinity for each round,the selected aptamers were cloned,sequenced and analyzed for their primaryand secondary structures.The 19 aptamers were divided into five groups based on primary sequence homology.Hairpin loop is the main motif in the predicted secondary structure and is supposed to be the binding part of the aptamers to CsA.The CsA-specific aptamers will be useful for enzyme-linked assays or immunofluorescence asses of CsA.
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87%. CONCLUSIONS The surveillance of overexpressing AmpC ?-lactamases in cefoxitin-resistant Gram-negative bacillus must be enhanced.The therapy of infections caused by related bacillus should make imipenem and meropenem a chief choice.DHA-1,CMY-2 and CMY-22 AmpC enzymes are found in Fuzhou.
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Objective To clone,express and identify the nuclear antigen Sm B′in E. coli to establish a new assay for detecting autoanti-body to Sm B′. Methods A full length cDNA of Sm B′was cloned from cell line HL-60 by RT-PCR. The PCR product was TA cloned and sequenced and inserted into the vector pGEX-5T. The recombinant plasmid was transformed into E. coli BL21. The positive clones were identified by restricted enzymes and induced by IPTG. The expression product was analyzed by SDS-PAGE and Western blot. Results The PCR product was about 700 bp in size which was in accordance with predicted 657 bp and sequencing result showed consistent with the sequence in GenBank. The pGEX-5T-Sm B′positive clone produced a 51 000 kD of fusion protein which was immunoreac-tive with anti-Sin B′confirmed by SDS-PAGE and Western blot. Conclusion The successful cloning and expression of nuclear antigen Sm B′laid a foundation for further research work.
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Objective To establish a new,rapid,simple and reliable assay for detecting autoantibody SSB.Methods A new dot immunogold filtration assay(DIGFA) was developed,in which the recombinant SSB protein expressed in Pichia pastoris was bound to nitrocellulose(NC) membrane and colloidal gold-labeled staphylococus protein A(SPA) was used as an indicator.Results The sensitivity and specificity of DIGFA were 100% and 98.75%,respectively.The agreement between DIGFA and ENA dot assay was 99.01%.Conclusion DIGFA for detecting autoantibody SSB is a good,rapid,simple and accurate assay for clinical diagnosis.
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Objective To establish a new assay for detecting autoantibody Jo-1, cloning and expressing human autoantigen Jo-1 in E.coli.Methods A full length cDNA of human autoantigen Jo-1 was cloned from cell line HL-60 by RT-PCR. The PCR product was TA cloned, sequenced and inserted into the carrier pGEX-5T.The recombinant plasmid was transformed into E.coli BL-21. The positive clones were identified by restricted enzymes and induced by IPTG. The expression product was analyzed by SDS-PAGE and Western blot.Results The PCR product was about 1 500 bp in size which was in accordance with predicted 1 526 bp and sequencing result showed the same with GenBank′s report.The pGEX-5T- Jo-1 positive clone produced a 75 000 fusion protein which had natural immunogenicity of human autoantigen Jo-1 by SDS-PAGE and Western blot.Conclusion Successfully cloning and expression of human autoantigen Jo-1 laid a foundation for further research work.