To construct expressive vector for human ScFv against core protein of hepatitis C virus (HCV core ScFv),and to express soluble HCV core ScFv in E.coli JM109. Using phage display technique, the recombinant phages were panned by recombinant core antigen which was coated in a microtiter plate, and after five rounds of biopanning, 86 clones were identified specific to core antigen. 750 bp fragment could be released from the plasmid of positive phage clones, and the sequence analysis indicated that we have obrained the ScFv DNA fragment. Then DNA fragment was inserted into the expressive vector pCANTAB5E, and E. coli host JM109 was transformed and induced by IPTG. The specificity of ScFv in the culture medium was evaluated by enzyme linked immunosorbent assay (ELISA).The molecular weight of expressed HCV core ScFv protein is 28 000 dalton as determined with the aid of SDS polyacrymide gel electrophoresis (PAGE). The expressed HCV core ScFv protein will be useful in the immunohistochemical study of liver tissue from patients with hepatitis C and gene therapy against HCV infection.

To screen and characterize human phage antibody to hepatitis C virus E2 antigen. The recombinant phages were panned by HCV E2 antigen which was coated in a microwell plate. After three rounds of biopanning, 56 clones specific to HCV E2 antigen were obtained. The specificity of scFv was determined by ELISA method and cross reaction with BSA and competitive inhibition assay. HCV E2 phage antibody had a specific combination character with recombinant hepatitis C virus E2 antigen. The DNA sequence data showed that the scFv coding gene was 771bp in size

In order to construct a prokaryotic expression vector of human receptor syt II N-fragment and to express recombinant MBP-Syt fusion protein in E.coli and to purify and identify its activity. According to codon preference of E.coli, a DNA fragment encoding human syt II N-fragment was synthesized, and then cloned into prokaryotic vector pMAL-c2x for sequencing. Then the recombinant plasmid pMAL-Syt was introduced into E.coli ER2566 by transformation for expression and the obtained engineered bacteria were induced by IPTG. The fusion protein was purified by amylose resin affinity chromatography and identified by SDS-PAGE and Western blot. The binding activity of the protein was determined by ELISA. It is concluded that MBP-Syt protein is of good binding activity.

Acute Disease ; Adult ; Humans ; Male ; Occupational Diseases ; diagnosis ; therapy ; Phosphines ; poisoning ; Poisoning ; diagnosis ; therapy

Objective:To express the B subunit of Shiga-like toxin type Ⅱ,and analyze its expression form and receptor-binding activity.Methods:The slt2b gene was obtained from EHEC O157∶H7 by PCR,and cloned to the expression vector pET22b(+).The genetically engineered bacteria pET22b(+)-stx2B/BL21 expressed the recombinant StxB after induced with IPTG.The renatured inclusion bodies were purified by ion exchange chromatography.The expression form of rStx2B was investigated by denaturing and native electrophoresis.The receptor-binding activity was confirmed by fluorescence detection and flow cytometer.Result:The constructed genetically engineered bacteria expressed the rStx2B at a high level.The purified protein was obtained after denaturation,renaturation and ion exchange chromatography.According to the denaturing and native electrophoresis,the rStx2B was expressed in a dimmer form,which consists of two monomers cross linked with disulfide bridge.The rStx2B showed good receptor-binding activity by Hela-binding assay.Conclusion:The genetically engineered bacteria were constructed successfully.The receptor-binding activity of rStx2B was independent of the pentamers.

OBJECTIVETo evaluate the efficacy and safety of testosterone undecanoate (TU) in the treatment of late-onset hypogonadism (LOH) by meta-analysis.

METHODSWe searched Pubmed (until April 1, 2014), Embase (until March 28, 2014), Cochrane Library (until April 17, 2014), CBM (from January 1, 2001 to February 2, 2014), CNKI (from January 1, 2001 to February 2, 2014), Wanfang Database (from January 1, 2000 to February 2, 2014), and VIP Database (from January 1, 2000 to Febru ary 2, 2014) for randomized controlled trials of TU for the treatment of LOH. We evaluated the quality of the identified literature and performed meta-analysis on the included studies using the Rveman5. 2 software.

RESULTSTotally, 14 studies were included after screening, which involved 1 686 cases. Compared with the placebo and blank control groups, TU treatment significantly increased the levels of serum total testosterone (SMD = 6.22, 95% CI 3.99 to 8.45, P < 0.05) and serum free testosterone (SMD = 4.35, 95% CI 1.86 to 6. 85, P < 0.05) but decreased the contents of luteinizing hormone (WMD = -2.23, 95% CI -4.03 to -0.42, P < 0.05), sex hormone binding globulin (WMD = 2.00, 95% CI 1.38 to 2.63, P < 0.05). TU also remarkably reduced the scores of Partial Androgen Deficiency of the Aging Males (WMD = -9.49, 95% CI -12.96 to -6.03, P < 0.05) and Aging Males Symptoms rating scale (WMD = -2.76, 95% CI -4.85 to -0.66, P <0.05) but increased the hemoglobin level (SMD = 2.35, 95% CI 0.29 to 4.41, P < 0.05) and packed-cell volume (SMD = 4.35, 95% CI 1.36 to 7.33, P < 0.05). However, no significant changes were shown in aspertate aminotransferase, alanine transaminase, prostate-specific antigen, or prostate volume after TU treatment (P > 0.05).

CONCLUSIONTU could significantly increase the serum testosterone level and improve the clinical symptoms of LOH patients without inducing serious adverse reactions. However, due to the limited number and relatively low quality of the included studies, the above conclusion could be cautiously applied to clinical practice.

Androgens ; therapeutic use ; Hemoglobin A ; metabolism ; Humans ; Hypogonadism ; blood ; drug therapy ; Luteinizing Hormone ; blood ; Male ; Prostate-Specific Antigen ; Randomized Controlled Trials as Topic ; Sex Hormone-Binding Globulin ; metabolism ; Testosterone ; adverse effects ; analogs & derivatives ; blood ; pharmacology

To produce antibodies capable of neutralizing botulinum neurotoxin type B(BoNT/B),We cloned the carboxy-terminal end of Hc containing the major determinants responsible for specific toxin,induced and purifed.The heavy-chain and kappa light-chain variable region gene repertoire of immunoglobulin were amplified individually from the spleen cell mRNA by RT-PCR and joined as a single-chain Fv(scFv)DNA fragment.These fragment were cloned into the phagemid pCANTAB5E and the phage display library was constructed.Results showed that the high affinity scFv was obtained after 4 rounds of panning,with its DNA sequence conforming to that of mouse antibody.

Objective To analyze the antimicrobial susceptibility of Mycoplasma isolates for rational antimicrobial therapy. Methods BioM?rieux IST kit was used for identification and susceptibility testing of Mycoplasma strains.Results Mycoplasma was positive in 49.5% of the specimens tested.Of the Mycoplasma detected,Ureaplasma urealyticum(Uu)alone accounted for 74.7%,Mycoplasma huminis(Mh)alone accounted for 18.1%,and Uu+Mh was identified in 7.2% of the patients.The results of antimicrobial susceptibility testing showed that the Mycoplasma isolates were most susceptible to doxycycline (98.1%).Ciprofloxacin was the least active (17.3%).Conclusions Doxyeycline,josamycin,and clarithromycin can be used in the treatment of urinary tract infections caused by Mycoplasma.

Objective To study the relationship between the intervals of Mitomycin C treatment and cytotoxicity, apoptosis and drug resistance for bladder cancer cells.Methods The bladder transitional cell cancer line BIU-87 was treated for two hours every time for five times with intervals of 24, 48, 72 and 96 hours respectively.Cytotoxicity was measured by MTT.p53,bcl-2,Bax and p170 expression were analyzed by Western blot.Results The IC_(50)(?g/ml)were 4.41,0.71,2.83,4.51and 6.16 with treatment intervals of 24, 48, 72 and 96 hours respectively, p53 and bcl-2 were significantly down-regulated and bcl-2/Bax was re- duced at 24 hour treatment interval but not changed at 48,72 and 96 hour intervals,p170 was not detected at 24 hour treatment interval but increasingly expressed at 48,72 and 96 hours intervals.Conclusion The in- terval of Mitomycin C treatment is closely related with cytotoxieity and apoptosis and drug resistance of blad- der cancer cells.The intervals of intravesical instillations may play an important role in the effect of chemotherapy.