ABSTRACT
Objective To construct the prokaryontic expression vector of the gene fragment which encodes the transpeptidase domain of penicillin binding protein 2a(PBP2a) of methicillin‐resistant Staphylococcus aureus(MRSA) ,and to express ,purify and i‐dentify the objective protein .Methods Strains of MRSA were isolated and identified from clinical samples ,according to the se‐quence of mecA gene recorded in GenBank ,the primers of mecA fragment which encoded the transpeptidase domain of PBP2a was designed .The gene fragment from MRSA was amplified by using polymerase chain reaction(PCR) and cloned into pET28a(+ ) plasmid .After being identified by enzyme digestion and sequencing ,the recombinant plasmid was transformed into the strain of Escherichia coli BL21(DE3)plysS .The expression of transpeptidase domain of PBP2a was induced by 0 .7 mmol/L IPTG ,the ex‐pressed products were purified by using Ni afinity chromatography ,then were analyzed by using Western blot .Results The recom‐binant expression vector was digested by BamHⅠ and EcoRⅠ ,and the products were at the expected size .The result of sequencing showed two bases undergoing mutation ,while there were no frameshift mutations .The expressed protein was identified by using SDS‐PAGE and Western blot ,a new protein band was visible at the relative molecular mass of 38 × 103 .Conclusion The corre‐sponding prokaryotic expression vector is successfully constructed ,and the transpeptidase domain of PBP2a is successfully ex‐pressed and purified .
ABSTRACT
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.
ABSTRACT
Objective To improve EP9-A2 for Bias estimation among multiple quantitative detection systems within full range of AMR.Methods 40 patients specimens were determined twice for serum total cholesterol by four detection systems(A,B,C and D).With system A served as comparative method,Bias between A and the other three was evaluated according to CLSI EP9-A2 separately.Furthermore,DD(distance from deviation to tolerable error)and its average confidence intervals between every two sys-tem were calculated and compared with zero.The confidence interval of greater than zero was served as criteria for accepting bias between every two system.Results Bias between A and the other three meet the analytical quality specification according to EP9-A2,although that of D system was positive,and those of B and C system were negative.DD between every two system obeyed nor-mality distribution.All biases between every two system wes acceptable except that between B and D,causing of their interval con-taining zero.After correcting of results from system D,Biases between every two system were all acceptable.Plots of confidence in-terval could provide a full range bias assessment within AMR.Conclusion Comparability and Bias estimation in full range of AMR for results between every two system among 3 or more systems could be evaluated by confidence intervals.
ABSTRACT
Due to the complicated and changeable clinical symptoms,and inexact pathogenesis,leukemia was facing huge challenges in diagnosis and therapy.Aptamer could be selected through CELL-SELEX.The aptamer not only display high affinity and specificity to target cell,but also it could discriminate the specific stage of cell,such as different classification,normal and tumor cells.So CELL-SELEX has great potention in the researches of leukemia.This paper systematically summarized the advantages of CELL-SELEX,the application in specific leukemia diagnosis,targeted therapy and the discovery of cancer biomarkers.(Chin J Lab Med,2013,36:377-379)
ABSTRACT
Myosin heavy chain 9 (MYH9)disorders are a group of ihherited thrombocytopenias resulted from the mutation of MYH9 gene,including May-Hegglin anomaly,Epstein syndrome,Fechtner syndrome and Sebastian syndrome.MYH9 disorders are very often misdiagnosed as idiopathic thrombocytopenic purpura (ITP).For better understanding of MYH9 of clinical and laboratory and getting enough attention in clinical practice,this review will focus on the pathogenesis,clinical manifestations,laboratory examination and differential diagnosis.
ABSTRACT
Aptamers are oligonucleotides or peptides those are able to bind tightly,by their specific three-dimensional shapes,to a variety of targets.Because of numerous merits ( high affinity,high specificity,small size,little immunogenicity,stable structures,and ease of synthesis),aptamers represent a valid alternative to antibodies and become a valuable research tool and show great application to fundamental research,drug selection and clinical diagnosis and therapy.The review describe the applications and the possible applications of aptamers in the diagnosis,treatment and pathogenesis of autoimmune diseases.
ABSTRACT
Objective To further research the biological functions of PES1,the ovarian SKOV3 cell line with inducible stable PES1 expression is established by using Tet-on system.Methods PES1 was cloned into pTRE-Tight vector via PCR and its expression was identified. After transfected the regulating plasmid pTet-on,SKOV3 cells were screened with G418 and re-transfected pTRE-Tight-PES1.The positive cell clones were screened out with hygromycin and were induced by doxycycline (Dox) to definite the best induction concentration.Growth velocity of SKOV3 cells stably expressing PES1 induced by Dox was detected with viola crystallina.Results The SKOV3 cells with inducible PES1 expression were screened out after the cells were transfected pTRE-Tight-PES1 constructed.Dox could dose-dependently induce the PES1 expression with the concentration under 2 mg/L,and 2 mg/L of Dox induced the highest PES1 expression.Growth velocity of SKOV3 cells transfected pTRE-Tight has no significant difference between the SKOV3 cells transfected nothing induced with Dox.However,the SKOV3 cells transfected pTRE-Tight-PES1 grew faster than the cells transfected pTRE-Tight or without transfection in the fourth day (P =0.001 ).Conclusion The inducible stable PES1 expression SKOV3 cells are successfully established and could be used to be an effective cell model to research the biological functions of PES1.The expression of PES1 could promote the growth of SKOV3 cells.
ABSTRACT
Objective To implement a real-time bidirectional communication function on Siemens StreamLab laboratory automation system, Methods Firstly, the system selecting sample was changed from the default settings and then the communication character string sent by the StreamLab was utilized by the laboratory information system to build up a real-time bidirectional control of the samples assay. Results New functions such as real-time signing in and real-time detection information getting of specimens could be performed by the StreamLab automatically.Conclusion The real-time bidirectional communication between laboratory automation system and laboratory information system can optimize the working flow and improve the work efficiency.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Aptazymes are a new artificial synzyme, selected from the random oligonucleotide sequence libraries against various of effector molecules. They own the advantages of an aptamer(the receptor site) and the ribozyme (the catalytic active site). Moreover, aptazymes as catalytic molecular beacon provide a new orientation for the quantitative analysis of effector molecules. Aptazymes not only have the application in genomics and proteomics, but also have potential applications in biosensor and DNA AND gate.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Systemic lupus erythematosus(SLE) is an autoimmune disease characterized by the production of autoantibodies against a wide spectrum of nuclear, cytoplasmic,and cell membrane autoantigens.Among them,the anti-Smith antibodies are specific for SLE and thus have been included as a criteria for diagnosis.They are not only a diagnostic marker but also a reliable measure of disease activity in SLE,we intend to describe the biological functions and clinical significance of the Smith antigen and the detection of anti-Smith antibodies.
ABSTRACT
Decoy receptor 3(DcR3),also known as tumor necrosis factor receptor 6B(TNFR6B) or TR6,is a newly discovered member of the tumor necrosis receptor superfamily,an apoptosis-prohibiting protein,which can both inhibit the apoptosis and promote the immunity escape of tumor cells.Recent researches have shown that DcR3 is expressed selectively in most human common tumors but not in normal adult tissue,and its up-regulation may predict the malignancy and prognosis of tumors.As a molecular marker of tumor,the high expression of DcR3 in the body fluid can help tumor detection and therapy monitoring.This novel biological technique can be used to inhibit the expression of the DcR3 gene and induce the apoptosis of tumor cells.
ABSTRACT
Decoy receptor 3,tumor necrosis factor receptor 6B or called TR6,is a newly member of tumor necrosis receptor superfamily.It plays very important roles in tumor immunity,autoimmunity,transplantation immunity,and anti-infection immunity by modulation of apoptosis and activity and differentiation of immunocytes.The current advance in molecular immunology of decoy receptor 3 was reviewed.
ABSTRACT
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.
ABSTRACT
Objective: To investigate the clinical value of a micro-array for multi-tumor marker detection (abbreviate C-12 system in the following) in four kinds of tumors (lung, liver, pancreas/colon and stomach cancers). Methods:30 lung cancer?19 liver cancer?24 pancreas/colon?22 stomach cancer and 173 non-tumor patients' serum were detected by C-12 system, and the results were analyzed by ROC curve.Results:There is no difference in the positive rate of single TM between C-12 system and the previously reports; The positive rate and the mean positive value of tumor patients were both obviously higher than that of non-tumor patients (P
ABSTRACT
Objective To implement a remote monitor system in a core medical laboratory.Methods Firstly,the Hub of StreamLab laboratory automation system was connected to a wireless router to build a wireless network,and several equipments were also integrated into the wireless network using wireless network cards.Secondly,VNC Server was installed into the control PCs of StreamLab LAS,CA7000 Coagulation Analyzer,DPC IMMULITE 2000 SMS and its chemiluminescence photometer,and remote control function of Dimension RxL Max was activated,then some control software such as VNC Viewer,Q/Remote and PC Anywhere were setup in two agency control computers.Finally,PCAnywhere and VNC Viewer were setup in the remote control computers in the Internet and Intranet,respectively.Results A remote control system with three functions was built successfully in a core medical laboratory.Conclusion The remote control system makes the daily operation and remote maintenance of the equipments easy.
ABSTRACT
Liquid chip,a new type of biochip classify encoding beads as carriers of reaction and signal detection,implement simultaneous determination protein,nucleic acid and other biomacromolecule in susp-ension liquid system.Apart from high-throughput characteristic as traditional biochip,the advantages of liquid chip are easy operation,the high-sensitivity,the high-accuracy,the high-precision and the great dynamic range and so on.It can be applied in the field of clinical laboratory diagnosis.In this review,we discuss the fundamental principle,detection,characteristics of liquid chip technique and the application in the field of clinical laboratory diagnosis.