OBJECTIVE To develop the strong and the specific multi-epitope antigen for the exploiting diagnosis of Treponema pallidum.METHODS The immuno-dominant epitopes of Tp0453 and Tp17 were amplified by PCR respectively,and subcloned into the expression vector pQE32 to generate multi-epitopes recombinant plasmid pQE32/Tp0453-17.The recombinant protein was expressed in Escherichia coli M15.The immunoresponse of recombinant fusion protein was analyzed by Western blot.RESULTS The multi-epitopes recombinant plasmid was successfully constructed,enzyme digestion analysis and sequencing showed that the inserted target genes were Tp0453 and Tp17 gene,compared with the gene reported by GenBank,it had 100% similarity;SDS-PAGE analysis showed the recombinant plasmid could be expressed in M15,its relatively molecular mass(Mr) of expressed product was about 52.0?103.The Western blot result showed the recombinant protein could be recognized by anti-T.pallidum positive serum.CONCLUSIONS The expressed multi-epitopes recombinant antigen showed excellent immunoresponse.The results lay the foundation for research on development of quick diagnostic kit applying to detection of T.pallidum infection.

Sepharose 6B was activated by epichlorohydrin in the strong base condition, and then reacted with solution of iminodiacetic sodium. The arms of IDA were conjuncted to the activated Sepharose 6B. Then the products were reacted with the solution of NiSO4. The arms of IDA were chelated with Ni2+,and the chelating resin―Ni2+-IDA could be prepared. The physicochemical indexes and performance in purifying protein of the expressing product were assayed with atomic absorption method and purifying aimed protein-human B lymphocyte stimulator(hBLyS) from the expressing products in E.coli. The results indicated that the performance of made gel is very good, and its price is less than 1/10 of that of commodity gel.

Objective To evaluate the value of a Treponema pallidum (TP) recombinant protein TP0993 in the serodiagnosis of syphilis.Methods A bioinformatics method was used to obtain the sequence of TP0993 gene.The open reading frame (ORF) without upstream non-coding region of TP0993 gene was ligated into the expression vector PET-28a (+),which was then transformed into Escherichia coli Rosetta.Isopropyl-β-d-thiogalactoside (IPTG) was used to induce the expression of TP0993 protein.The expressed protein was purified with nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography.Western blot was performed to evaluate the immunoantigenicity of the protein.New Zealand rabbits were immunized with the recombinant protein for immunogenicity evaluation.Indirect enzyme linked immunosorbent assay (ELISA) was developed by using the purified recombinant protein to coat microwell plates.Anti-TP antibodies were detected by the established ELISA and TP particle agglutination assay (TPPA) in 480 clinical serum samples.Results The prokaryotic expression vector PET-28a (+)-0993 was successfully built,and a fusion protein with a relative molecular weight of about 34 000 Da was attained after IPTG-induced expression and purification.Western blot proved that the recombinant protein could specifically react with clinical sera positive for anti-TP IgG antibodies.Specific humoral response was elicited in New Zealand rabbits by the recombinant protein.Compared with TPPA,the established indirect ELISA showed a sensitivity of 88.3％ and a specificity of 85.8％.There was a consistency of 86.5％ between the indirect ELISA and TPPA.Conclusion The expressed recombinant protein showed favorable immunocompetence,and may serve as a candidate antigen for serodiagnosis of syphilis.

To investigate the effects of human anti-BAFF scFv-Fc against the hsBAFF, ICR mice were randomly divided into six groups: control, hsBAFF (1 mg x kg(-1)), hsBAFF (1 mg x kg(-1)) + Ab (1 mg x kg(-1)), hsBAFF (1 mg x kg(-1)) + Ab (2 mg x kg(-1)), hsBAFF (1 mg x kg(-1)) + human IgG (1 mg x kg(-1)) and hsBAFF (1 mg x kg(-1)) + human IgG (2 mg x kg(-1)) groups. The effects of scFv-Fc administration on the proliferation of B lymphocytes were evaluated using an MTT assay. The titres of antibody in the serum and B lymphocytes differentiation were assessed by ELISA and flow cytometry, respectively. The results showed that administration of scFv-Fc to mice injected with hsBAFF significantly prevented human BAFF-induced increases in splenic B cell numbers and serum immunoglobulin levels. Furthermore, this fully human antibody would avoid inducing the human anti-mouse antibody (HAMA) response when used in humans. These findings suggest that the compact antibody may be useful in therapeutic or diagnostic application of the BAFF-associated autoimmune diseases in human.

The antibacterial peptide CM4 having potent antifungal activity on inhibitiong the cell wall regeneration of Saccharomyces cerevisiae protoplasts.When the peptide increased,the ratio of the regenerated colonies drop obviously.To study the antifungal mechanism of the antibacterial peptide,fluorescence\|labeled peptide mixted with the protoplast of yeast,then confocal laser scanning microscopy were performed.The results indicated that the peptides interactted with the protoplast membrane and damaged the structure of the membrane,then the permeation of protoplast changed.Finally the protoplasts with the peptide failed to regenerate the cell walls leading to killing the cell.

OBJECTIVE To evaluate the spectrum of imipenem-resistant Pseudomnas aeruginosa and the production of metallo-?-lactamase.METHODS The clinical strains of P.aeruginosa were collected from Jan to Dec 2007.The results of antimicrobial susceptibility tests and detection of metallo-?-lactamase were analyzed.Antimicrobial susceptibility tests were performed by K-B methods;the production of metallo-?-lactamase was tested by CAZ-EDTA synergy method.RESULTS Sixty strains were isolated,imipenem-sensitive and resistant strains were 40(66.7%) and 20(33.3%),respectively,and 7 strains with metallo-?-lactamase were detected.Among imipenem-resistant strains,at least 90.0% strains were resistant to meropenem,gentamicin,tobramycin,ciprofloxacin and SMZ-TMP;at least 80.0% strains were resistant to piperacillin and piperacillin/tazobactam;50.0% strains were resistant to ceftazidime and cefepime;polymyxin E was less resistant than others.Twenty strains were resistant to at least 3 antimicrobial agents,which was obviously higher than 27.5% of imipenem-resistant strains.CONCLUSIONS The resistance rate of imipenem-resistant P.aeruginosa is higher than imipenem-sensitive ones.The production of metallo-?-lactamase is one of the mechanisms of P.aeruginosa resistance to imipenem and shou1d be detected carefully,which could help us medicate reasonably in clinic and avoid using medicine which could induce and strengthen the resistance.

OBJECTIVE: To evaluate the curative effect and clinical significance of bone cement on coagulation functions during percutaneous vertebroplasty in patients with osteoporotic spinal compression fractures.METHODS: A total of 24 patients, comprising 18 females and 6 males, aged 69 years averagely (range 48-83 years), with 44 osteoporotic vertebral compression fractures underwent percutaneous vertebroplasty in Department of Spinal Surgery, Third Hospital of Hebei Medical University between December 2006 and December 2007. The fracture segment was within T_5-L_3 (20 thoracic vertebrae and 24 lumbar vertebrae). Under the guidance of C-arm fluoroscopy, bone marrow biopsy needle was inserted percutaneously via transpedicular way into the fractured vertebrae. Polymethylmethacrylate (PMMA, bone cement) was injected into the fractured vertebrae. The relative parameters were observed in all patients 10 minutes before, 10 minutes, 30 minutes, 1 hour, 2 hours and 3 hours after bone cement implantation, including prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (FIB), plasma protamine paracoagulation test (3P test), and D-dipolymer (D-D). RESULTS: PT was decreased, and FIB, 3P test, D-D were increased 10 minutes after bone cement implantation in percutaneous vertebroplasty peaked at 1 hour and gradually decreased afterward; moreover, there were significant difference between bone cement preimplantation and 10 minutes, 30 minutes, 1 hour, 2 hours and 3 hours after bone cement implantation (P < 0.05), but no difference was observed in APTT and TT (P > 0.05). The influence of bone cement on the parameters was vanished in 3 hours after bone cement implantation, and all indexes were similar to pre-implantation (P > 0.05).CONCLUSION: Bone cement implantation causes temporal hypercoagulabale state in percutaneous vertebroplasty. It is important to monitor blood clotting state in 3 hours after bone cement implantation in order to avoid thrombus disease.

Objective Toinvestigatetheriskoflong-termrecurrenceofstrokeanditspredictorsin youngpatientswithischemicstroke/transientischemicattack(TIA).Methods Theclinicaldataofthe consecutive young patients (18-45 years)with ischemic stroke/TIA (within 2 weeks after onset)admitted to the department of neurology in the First Affiliated Hospital of Sun Yat-Sen University between August 2008 and July 2013 were enrolled prospectively. All patients were regularly followed up for a long time (The patients were followed up at the 1 st,6 th,and 12 th month after onset;then they were followed up once for every 6 months)in order to investigate stroke recurrence. The Kaplan-Meier curves were used to analyze the cumulative stroke recurrence rate of all patients. The last contact time for patients lost to follow was used as censored data to be enrolled in the analysis. The univariate analysis of the related risk factors for stroke recurrence using Log-rank test. Multivariate Cox proportional hazard regression was used to detect the related risk factors associated with stroke recurrence (adjusting for age and sex). The variables of the results of Log-ranktestP≤0.1wasselectedandenrolledinthemultivariateregressionanalysis.Results Atotalof 312 patients were enrolled in the analysis,including 294 with ischemic stroke and 18 with TIA. Their mean follow-up time was 34 ± 19 months. Thirty-four patients had recurrent stroke,including 23 with ischemic stroke,7 with TIA,and 4 with cerebral hemorrhage. The cumulative recurrence rates of stroke at 1 ,3 , and 6 years after onset were 6. 2%,10. 3%,and 16. 4%,respectively. The results of multivariate Cox proportional hazards regression analysis showed that hypertension (risk ratio [RR]2. 159;95% confidence interval [CI]1. 048-4. 447,P=0. 037)and cardioembolism (RR,2. 869;95%CI 1. 119-7. 357,P=0.028)weretheindependentpredictorsforstrokerecurrence.Conclusion Theoverall6-yearriskof recurrent stroke is not high in the Chinese young patients with ischemic stroke/TIA,but the risk of stroke recurrence is relatively higher in the first year. Hypertension and cardioembolism are the potential predictors of stroke recurrence;therefore,attention should be paid in clinical practice.

Objective To investigate the risk factors, diagnostic evaluation, etiology, and treatment in young patients with ischemic stroke. Methods The clinical data of young patients (age range 18 - 45 years) with ischemic stroke (within 2 weeks of stroke onset) admitted to the Stroke Center, the First Affiliated Hospital of Sun Yat-sen University from August 2008 to July 2013 were registered prospectively. Results A total of 300 patients were enroled, their age was 37. 8 ± 6. 8 years. There were 227 males (75. 7% ). The age of 84 patients (28. 0% ) was ≤35 years. The most common risk factors were smoking (43. 3% ), hypertension (38. 7% ), and hypercholesterolemia (38. 0% ). The positive result detection rates were lower in the antinuclear antibody (8/278), anticardiolipin antibody (34/250), 24-h Holter monitoring (2/60 ), and transthoracic echocardiography (38/232). According to the MRI findings, the detection rate of the white matter changes, old infarcts, single acute infarcts, and multiple acute cerebral infarcts were 40/282, 77/282, 145/282, and 137/282, respectively. The stroke subtypes of TOAST: large artery atherosclerosis (26. 7% ), smal artery occlusion (18. 0% ), cardioembolism (10. 0% ), other determined etiology (23. 0% ), and undetermined etiology (22. 3% ). Large artery atherosclerosis was mainly involved in anterior circulation (69/80) and intracranial arteries (75/80). In other definite causes, cerebral artery dissection was most common (36/69), and other causes included moyamoya disease (7/69) and infectious vasculitis (7/69), etc. The average length of hospital stay was 15. 4 d. The mean National Institutes of Health Stroke Scale score on admission was 7. 0, and the mean modified Rankin scale score at discharge was 2. 5. The complication rate during hospitalization was 9. 7% . 80. 3% and 48. 3% of patients received anti-platelet drugs and statins therapy during hospitalization.Conclusions This study used a prospective single-center method. It conducted a comprehensive analysis of risk factors, diagnostic evaluation, treatment, and etiology in current Chinese young patients with ischemic stroke. Its research data wil provide useful information for establishing a diagnostic strategy of high performance cost ratio, in-depth understanding of its pathophysiological mechanisms, and improving treatment strategies in Chinese young patients with ischemic stroke.

Objective To clone, express Tp0319 gene from Treponemapallidum (T. pallidum), and to assess the immunocompetence of recombinant protein. Methods The immuno-dominant region of Tp0319gene was chosen by computer analysis, amplified from T. pallidum complete genome by PCR, subcloned into the expression vector pQE32 to construct a recombinant plasmid, pQE32/Tp0319, which was then expressed in E. coli M15. The recombinant protein was purified with Ni-NTA affinity chromatography, and identified by using sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blot. New Zealand rabbits were immunized with the recombinant protein, and the titer of anti-Tp0319 antibodies in sera from immunized rabbits were measured with indirect ELISA. Also, indirect ELISA with the recombinant Tp0319 as coating antigen was performed to detect the anti-Tp0319 antibody in sera from 200 normal human controls and 200 patients with syphilis. Results The prokaryotic expression vector pQE32/Tp0319 was constructed successfully, and the recombinant protein Tp0319 with a molecular weight of about 30 000 was attained. Specific humoral response was elicited by the recombinant protein in New Zealand rabbits and the specific antibody titer was more than 1: 10 240 after immunization for 3 times. Western blot proved that the recombinant protein could specifically react with anti-T. pallidum IgG antibody-positive sera. Indirect ELISA was successfully developed with the recombinant Tp0319, and detected antibodies to T. pallidum in control sera with a sensitivity and specificity of 100% (40/40), respectively. Compared with T. pallidum particle agglutination (TPPA) assay, the sensitivity and specificity of the indirect ELISA were 92.6% and 100%, respectively, in the detection of T. pallidum in sera from patients and controls, and the concordance between the indirect ELISA and TPPA was 96%. Conclusions The prepared recombinant protein shows a satisfactory immunocompetence, which may lay a foundation for its further application in the serodiagnosis of syphilis.