Objective To explore the establishment of peptide mapping database of Candida albicans ,laying the foundation for rapid diagnosis of Candida albicans infection .Methods 96 Candida albicans were collected clinically ,and its DNA was extracted . Polymerase chain reaction(PCR) was used to amplify the ITS1-5 .8S-ITS2 gene fragments and restriction endonucleases were a-dopted to identify them .Surface enhanced laser desorption ionization-time of flight-mass spectrometry(SELDI-TOF-MS) instrument was applied to detect the Candida albicans peptide mapping ,and Ciphergen ProteinChip software was used to collect data automati-cally .The established peptide mapping database was verified by confirmed Candida .Results According to restriction fragment length polymorphism analysis ,96 strains were confirmed as Candida albicans .15 peptide peaks were captured by SELDI-TOF-MS chips .Five peptide peaks of them with stable expression were screened out ,and the similarity analysis software was used to estab-lish peptide mapping database of Candida albicans .More than 95% of similarity was found between peptide mapping of Candida albicans and established database ,while less than 50% was found between peptide mapping of other Candida species and database . Conclusion The establishment of peptide mapping database of Candida albicans provides a theoretical basis for the rapid diagnosis of Candida albicans infection .

Objective To investigate SCCmec types in clinical isolates of methicillin-resistant Staphylococcus epidermidis (MRSE) carrying psm-mec.Methods We collected 165 strains of Staphylococcus epidermidis identified by automated microbiological identification system and screened MRSE by PCR amplification of esp and mecA gene.Strains with psm-mec were identified by amplification of psm-mec,fudoh and p221 DNA fragment;mec,ccr and SCCmec typing was conducted by multiplex PCR assay.Results Among 138 strains of MRSE,29 strains were identified as MRSE with psm-mec,and the carrying rate was 17.58%.Results of mec and ccr typing by multiple PCR showed that MRSE with psm-mec carried Class A mec,but the ccr type had obvious diversity.Results of SCCmec typing showed that all strains with psm-mec belonged to type Ⅱ and/or Ⅲ SCCmec.Conclusion Clinical isolates of MRSE with psm-mec carry homologous type Ⅱ and/or Ⅲ SCCmec harboring Class A mec.

Objective To establish protein fingerprinting identification model of Pseudomonas aeruginosa (P. aeruginosa) and to lay a foundation for rapid identification of P. aeruginosa by proteinchip golden array. Methods Sixty-four P. aeruginosa and one hundred and ninety-nine control bacteria identified in our laboratory were collected and divided into training and testing group. Surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) and proteinchip golden array were used to detect the protein profiling of the bacteria. Data were automatically collected by Ciphergen Proteinchip Software and protein markers of P. aeruginosa were screened by BioMarker Wizard Software. Classification tree model was developed and validated by BioMarker Patterns Software. The model was blindly tested with twenty-nine P. aeruginosa and sixty-four control bacteria. Results Eighty protein peaks were detected between 3000 and 20 000, among which fifty-eight ones showed significantly difference between P. aeruginosa and the control bacteria (P＜0.01). By BioMarker Patterns Software, one protein peak ( M/Z at 14 045.2) was chosen to develop a classification tree model. The results exhibited with sensitivity of 96. 55% and specificity of 100%. Conclusion Proteinchip golden array has the potential for rapid identification of P. aeruginosa.

Objective To establish protein fingerprints of common bacteria in clinics and to lay a foundation for rapid identification of bacteria.Methods Strains of Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa and Staphylococcus aureus were detected by surface enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS).Stable expression protein peaks were screened and the data was input into the self-constructed Fingerwave software for identification of target bacteria by protein fingerprint comparison.Two hundred and fifty-six clinical isolates,including E.coli,K.pneumoniae,P.aeruginosa and S.aureus were detected and the data was compared with constructed database to evaluate its diagnostic value.Results The protein fingerprints including four common bacteia was used to identify the target bacteria with identification rate of 93.1％ (54/58) for E.coli,87.2％ (75/86) for K.pneumoniae,96.2％ (60/63) for P.aeruginosa and 96.2％ (51/53) for S.aureus,respectively.Conclusion Common bacteria can be rapidly identified by using the protein fingerprint comparison,which provides a powerful tool for bacterial identification.

Objective To investigate the mechanism of one carbapenems resistant Raoultella planticola( R.planticola) isolate.Methods This is an experimental study.R.planticola was isolated from a patient′s drainage fluid from orthopedic department in November 2010 in Sichuan Provincial People′s Hospital.Minimum inhibitory concentration of R.planticola to 13 antibiotics was determined by using the agar dilution method.Modified Hodge test was used to detect carbapenemase .EDTA synergistic test was performed to research metallo-beta-lactamase.The genes coded the β-lactamase were amplified by polymerase chain reaction ( PCR ) , including class A carbapenemase ( KPC ) , class B carbapenemases (NDM, IMP, VIM, SIM), extended spectrum beta-lactamases[ESBL(CTX, TEM, SHV)], and AmpCβ-lactamases ( FOX, EBC, ACC, DHA, CIT, MOX).Results The susceptibility test showed that R.planticola was resistant to 9 antibiotics.MIC value of meropenem for R.planticola was up to 32 mg/L.R.planticola kept intermediary to imipenem , whereas it was susceptible to cefepime , amikacin and polymyxin B.Modified Hodge test and EDTA synergistic test were positive in R.planticola.Class B carbapenemase (IMP) gene and two extended spectrum β-lactamases(CTX, SHV) genes were positive by PCR.The genes were conformed as IMP-4, CTX-M3 and SHV-12 by sequencing and compared with GenBank.Other resistant genes were negative.Conclusion IMP-4 was identified in R.planticola, the combined produce IMP-4 and ESBLs might be the main mechanism of R.planticola resistant to carbapenems.

Objective To search for protein markers in urine from patients with diabetic nephropathy by proteomic method and discuss its clinical significance in laboratory diagnosis of diabetic nephropathy. Methods This study included 129 patients with diabetic nephropathy, 61 diabetes mellitus patients, and 102 healthy volunteers. The urinary protein profiles were obtained using surface-enhanced laser desorption-ionization time of flight mass spectrometry (SELDI-TOF-MS) and Au Chip (ProteinChip Gold Array). The differential peaks were screened by Biomaker Wizard software and the decision tree pattern was developed by Biomarker Patterns Software (BPS). The model was blindly tested to validate diagnostic efficiency. Some differentially expressed protein was preliminarily identified according to the molecular weight as compared with mass spectrometry data of standard proteins. Results Totally 40 distinguished protein peaks(t value: - 9.81-24.52, P < 0.05) were obtained after comparing the samples between diabetic nephropathy and the control groups. The peak with m/z 66 916 was automatically screened by BPS to develop decision tree pattern. The pattern was blindly tested and yielded a sensitivity of 98.7% (78/79) and a specificity of 98.2% (111/113). After we compared results from diabetic nephropathy with those from diabetes mellitus, twenty-four differential peaks were obtained in diabetic nephropathy (t value: -6.95-14.45,P < 0.05). The peaks with m/z 4 008, 11 619 and 66 916 were automatically screened by BPS to establish decision tree pattern. The model was blindly tested and yielded the sensitivity(129/129) and specificity(61/61) of 100%. After we compared our results with mass spectrometry data of standard proteins, the four differentially expressed proteins with m/z 11 619, 23 529, 66 916 and 79 378 were supposed to be β_2-microglobulin, α1-microglobulin, albumin and transfcrrin. Conclusion The preliminary results suggest that these SELDI-TOF and Au chip have the potential application value in identification of protein source and early diagnosis of diabetic nephropathy, and evaluation of renal injury.

Objective To investigate the SCCmec types of clinically isolated methicillin‐resistant Staphylococcus epidermidis (M RSE) .Methods Eighty‐four strains of clinically isolated Staphylococcus epidermidis identified by the fully automatic microbio‐logical identification system were collected and performed the MRSE identification by PCR for amplifying esp and mecA genes and SCCmec typing .Its distribution characteristics were analyzed .Results Esp gene was amplified in 84 strains and the detection rate of mecA was 76 .19% (64/84) ,in which the MRSE detection rates in blood ,sputum ,urine and wound secretion were 76 .8% , 68 .8% ,100% and 71 .4% respectively .The multiple PCR amplification displayed that among 64 strains of MRSE ,19 strains were SCCmec simple type ,in which 19 strains were SCCmec type Ⅰ and 3 strains were SCCmec type Ⅲ ;42 strains were SCCmec mixed type ,in which 2 strains were SCCmec mixed type Ⅰ and Ⅱ ,14 strains were SCCmec mixed type Ⅰ and Ⅲ ,12 strains were SCCmec mixed type Ⅰ ,Ⅱ and Ⅲ ,5 strains were SCCmec mixed type Ⅱ and Ⅲ ,a strains were and SCCmec mixed type Ⅲ and Ⅳ .Conclu‐sion The SCCmec type in clinically isolated MRSE shows obvious diversity and its majority is SCCmec mixed type .

Objective To discuss the application value of modified Hodge test(MHT) for screening carbapenemase-producing Enterobacteriaceae.Methods The 24 Enterobacteriaceae reduced susceptibility to carbapenems were detected by MHT.At the same time,polymerase chain reaction(PCR) was used to detect carbapenemase genes of KPC,NDM,IMP,SIM and VIM.PCR products were sequenced and the results were compared with the sequences of Gen Bank database.Comprehensive analysis the application value of MHT and PCR to detect carbapenemase.Results Among these 24 strains,13 stains appeared to produce carbapenemase by MHT,5 positive strains were found to carry carbapenemase genes by PCR.By comparing with the sequences of Gen Bank database 1 strain were confirmed to KPC-2 and 4 strains were confirmed to IMP-4.We found that 4 strains of Enterobacteriaceae,detected carbapenemase by MHT and PCR at the same time.9 strains of MHT were positive,but we couldn′t detect the carbapenemase genes.1 strain of MHT was negative,but carbapenemase gene was found in the strain.Conclusion The value of MHT to screen carbapenemase-producing Enterobacteriaceae is necessary to further study.

Objective To investigate the genetic location of SCCmec-associated psm-mec in Staphylococcus hominis isolated from blood culture,and to lay a foundation for further functional studies of psm-mec in Staphylococcus hominis.Methods 25 strains of Staphylococcus hominis isolated from positive blood culture were collected.mecA and psm-mec gene were amplified by PCR,and the SCCmec types were determined by the results of multiplex PCR assay.For analyzing the genetic location characteristic of psm-mec in SCCmec,three pair special PCR primers were used to measure mecR1/psm-mec,psm-mec/xylR and fudoh respectively.Results There were 21 strains of methicillin-resistant Staphylococcus hominis and 4 strains of methicillin-sensitive Staphylococcus hominis. The positive rate of psm-mec gene in methicillin-resistant Staphylococcus hominis was 47.6%,and no psm-mec gene was found in methicillin-sensitive Staphylococcus hominis.Among psm-mec positive strains,2 strains belonged to SCCmecⅢ,5 strains belonged to SCCmecⅢ-like,and 3 strains belonged to new SCCmec types.All of the 10 psm-mec positive strains were mecR1/psm-mec,psm-mec/xylR and fudoh gene positive.Conclusion SCCmec-associated psm-mec extensively exists in methicillin-resistant Staphylococ-cus hominis isolated from positive blood culture,which distributes mainly in typical SCCmecⅢ,SCCmecⅢ-like and new SCCmec types and locates between mecR1 and xylR gene.

To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.
Base Sequence ; Cloning, Molecular ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Genetic Vectors/genetics ; Hep G2 Cells ; K562 Cells ; Molecular Sequence Data ; Receptors, Transferrin/*immunology ; Recombinant Fusion Proteins/biosynthesis ; Recombinant Fusion Proteins/genetics ; Single-Chain Antibodies/*biosynthesis ; Single-Chain Antibodies/genetics