Objective:To obtain the functional single chain Fv antibody(scFv) against acidic isoferritin(AIF).Methods:An expression vector pPOW4c9 was constructed by subcloning AIF4c9 scFv gene into a heat-inducible bacterial expression plasmid pPOW3. Then recombinant vector was introduced into E.coli DH5? by electro-transformation. The soluble expression was performed by temperature induction. After purified by the Ni-chelating chromatography, the recombinant anti-AIF scFv was characterized.Results:Soluble expression of the scFv in E. coli was achieved. The yield of purified anti-AIF scFv was 1.6 mg/L. The recombinant protein recognized AIF specifically identified by ELISA and western blotting, and an affinity constant of scFv was 3.18?10~ -8 mol/L.Conclusion:The results indicate that recombinant soluble scFv retains the specific binding activity to AIF.

Objective To establish a new effective method by using real-time polymerase chain reaction (PCR) to detect single nucleotide polymorphism (SNP) typing for rapid identifying apolipoprotein E alleles.Methods To determine alleles of human apolipoprotein E genetic polymorphism at Cys112Arg locus was detected by PCR melting curve analysis with fluorophore SYBR Green I. In order to increase the speciality of SNP assays, high fidelity Taq polymerase was used. The reliability of SNP typing was validated by comparison with the results of direct DNA sequencing.Results Each sample was determined by double tubes, and two melting curves were analysis. As compared the Tm value of samples with the Tm of standard substance, the apoE genotype of samples was determined. The apoE genotype of 30 samples were E3/3 (27/30) and E3/4 (3/30) respectively, which was accordant with the results of PCR-RFLP and DNA sequencing.Conclusion The presented allelic assay was specific, easy to operate and applicable for discrimination of apolipoprotein E genotyping of human blood.

Objective To study the immune regulatory effect of natural killer cell(NKT) in the early stage of murine liver injury induced by type 5 adenovirus(Ad5). Methods Animal models were con-structed by injected C57BL/6 mice with 1.5×109-3×109 PFU Ad5 into the tail vein. Liver injury of mouse at day 0, 1, 2, 3, 4, 5 after infection was determined by HE staining and serum ALT/AST(alanine amin-otransferase/aspartate aminotransferase) level. Flow cytometry analysis was used to measure the proportion of lymphocytes, expression of Fas/FasL on the surface of NKT cells and level of IL-4, IFN-γ/in NKT cell plasma in the infected mouse liver. RT-PCR was applied to semi-quantify the chemokines and their receptors mRNA in infected mouse liver. Results NKT cells of mouse increased significantly at day 1 after infected with high titer Ads(3×109 PFU), expression of FasL on NKT cell and plasma IL-4, IFN-γ/level in NKT cells were also up-regulated, hence the obviously infiltration of lymphocytes in routine liver. Comparing with high titer Ads infection, low titer Ads infection (1.5×109 PFU) lead to little change of NKT cell proper-tion, and fewer infiltration of lymphocytes in murine liver. Hepatic chemokine RANTES, 1P-10, and MIP-1β mRNA expression in C57BL/6 was up-regulated 2 d after intravenous administration of 3×109 PFU Ad5. Corresponding chemokine receptor CCR5, CCR1, CXCR3 mRNA expression was up-regulated 3 d after in-fection. Conclusion NKT cells play an important role in lymphocytes recruitment into the liver of mouse in-fected with AdS, which may relate to up-regulatio of the plasma IL-4, IFN-γ level and expression of FasL of NKT cells, therefore facilitating the production of chemokines, e.g. IP-10 and Mig.

OBJECTIVE To analyze the distribution and anti-microbial resistance profile of pathogenic bacteria isolated from hospitalized neonates in Wuhan area.METHODS The strains of bacteria isolated from neonates in hospitalization of Wuhan area from 2006 to 2007 were identified by VITEK 32 automatic bacteria system and antimicrobial susceptibility test was performed by Kirby-Bauer method.The standards of NCCLS issued in 2006 were used to assess the results of antimicrobial susceptibility.RESULTS A total of 3892 strains of bacteria were isolated from 10053 specimens,the positive rate was 38.7%.The Gram-positive cocci and Gram-negative bacilli accounted for 41.0% and 55.2%,respectively.The leading 8 species on the list of isolated strains in the two years accounted for 90.8% of all the isolated strains.MRCNS accounted for 68.6% of coagulase negative staphylococcus,and the MRSA kept a low rate(2.6%) in Staphylococcus aureus.The infection rate of newborn by Enterococcus in blood was remarkably higher than that of the past report in this area.The ?-lactamases-producing Escherichia coli and Klebsiella pneumoniae were accounted for 46.9% and 34.7%,respectively.CONCLUSIONS The distribution and anti-microbial resistance of hospitalized neonates in Wuhan area have regional and group characteristics;it′s necessary to strengthen monitoring the regional epidemic characteristic status and the drug-resistant status of the pathogenic bacteria among neonates.