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 investigate the HBV quasispecies groups in the patients with chronic HBV infection. A set of specific primers was synthesized according to HBV DNA sequence of the Chinese strain, and the whole pre core/core region was amplified by PCR method from the serum of 4 patients with chronic HBV infection.Then the PCR products were subcloned into pGEM Teasy vectors. Clones were selected to be sequenced,and then sequence comparison was made to find the difference. Each sequence of selected clones was found to be different. The homology of the nucleic acid sequence from the same patient was above 98 0%. There were many mutation types in this region, including point substitution, core region internal deletion, as well as frame shift reading. The results indicated that there were HBV quasispecies groups in chronically infected patients. The mutation in this region may influence the prognosis of HBV infection.

T7 cDNA phage display method was employed to find the binding protein of PreSl protein. PreSl protein was coated in a 96-well ELISA plate, and then T7 cDNA library phages were bound to the target protein. Phages which did not bind to garget protein were washed away and the binding phages were eluted. Insertions from different clones were sequenced, and the deduced amino acid sequences were analyzed by Vector 6.0 software. Using BLAST software in GenBank, whole length of amino acid sequence of binding protein was obtained. After 4 rounds of biopanning, recombinant T7 phages with binding ablity were amplifed by infection to E. coli. One piece of amino acid sequence was found to be amino terminal of product of glioma tumor suppressor candidate region gene 2 (GLTSCR2). There was a binding domain KxPxKSGxxxL in these clones. T7 cDNA phage display technique can be used bo find the ligand. GLTSCR2 coding protein may be the binding protein to preSl protein of HBV.\;

Alzheimer's disease(AD)and Parkinson's disease(PD)are common neurodegenerative diseases that seriously threaten the health of the elderly, involving various abnormalities in physiology and metabolism including the impairment of mitochondrial function, Ca 2+ homeostasis deregulation, oxidative stress, aggregation of misfolded proteins, autophagy and inflammation.However, mechanisms underlying the pathogenesis of these diseases have not been clearly elucidated, thus impeding advances in their treatment.In recent years, it has been found that mitochondria-associated endoplasmic reticulum membranes(MAM)play an important role in the development of AD and PD and exert their effects by regulating the functions of mitochondria and endoplasmic reticula.This article reviews studies of the past decade related to the effects of MAM on AD and PD, aiming to generate insights for exploring the constituent proteins of MAM and the molecular mechanisms of AD and PD via endoplasmic reticulum-mitochondria Ca 2+ transport and ER stress regulated by MAM.

Objective:To investigate the expression pattern of microRNA-146a in Brucella patients and its correlation with antibody titers.Methods: By using real time PCR assay, expression levels of microRNA-146a in sera samples from 20 brucellosis patients and 20 healthy volunteers were analyzed.The correlation between expression level of microRNA-146a and serum antibody titers were analyzed with SPSS17.0.Results: A quantification curve of microRNA-146a was constructed with synthesized standard.Expression levels of microRNA-146a among brucellosis patients were significantly lower than those in 20 healthy volunteers (P<0.001).For brucellosis patients,the expression level of microRNA-146a was negatively related with antibody titers (P<0.05). Conclusion:Expression of miRNA-146a in brucellosis patients was significantly inhibited and negatively related with antibody titer.

Wnt signaling is implicated in the control of cell growth and differentiation during neural stem cell(CNS) development.Wnt3a, one of wnt gene family members, has effect on regeneration neurospheres and differentiation into neurons.Wnt3a inhibits regeneration of neurospheres, and promotes its differentiation. In vitro neurosphere was cultured in a serum-free defined medium DMEM/F12 supplemented with bFGF and EGF. Dissociated cells were plated onto poly-d-lysine-coated coverslips and propagated in medium containing recombined Wnt3a-adenovirus. Plenty of Nurr1 were detected by RT-PCR after 3 days. Wnt3a combined AA would improve NSC differentiation into dopaminergic (DA) neuron. The quantity of DA neuron is obviously more than the AA alone group's. Moreover, the expression of TH mRNA is 1.86 fold in Wnt3a combined AA group. Induced cells were immunostained for TH and DAT. The proportion of TH-positive was (37.42 ? 2.54) % (P

OBJECTIVETo search for genomic DNA sequence of the augmenter of liver regeneration (ALR) of rat.

METHODSPolymerase chain reaction (PCR) with specific primers was used to amplify the sequence from the rat genome.

RESULTSA piece of genomic DNA sequence and a piece of pseudogene of rat ALR were identified. The lengths of the gene and pseudogene are 1508 bp and 442 bp, respectively. The ALR gene of rat includes 3 exons and 2 introns. The 442 bp DNA sequence may represent a pseudogene or a ALR-related peptide. Predicted amino acid sequence analysis showed that there were 14 different amino acid residues between the gene and pseudogene. ALR-related peptide is 84 amino acid residues in length and relates closely to ALR protein.

CONCLUSIONThere might be a multigene family of ALR in rat.

Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; DNA ; genetics ; Female ; Humans ; Liver Regeneration ; genetics ; Male ; Mice ; Molecular Sequence Data ; Polymerase Chain Reaction ; Proteins ; genetics ; Pseudogenes ; genetics ; Rats

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is considered a serious global threat. However, little is known regarding the multidrug resistance (MDR) mechanisms of CRKP. This study investigated the phenotypes and MDR mechanisms of CRKP and identified their clonal characteristics. METHODS: PCR and sequencing were utilized to identify antibiotic resistance determinants. Integron gene cassette arrays were determined by restriction fragment length polymorphism (RFLP) analysis. Multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for epidemiological analysis. Plasmids were typed by using a PCR-based replicon typing and analyzed by conjugation and transformation assays. RESULTS: Seventy-eight strains were identified as resistant to at least one carbapenem; these CRKP strains had a high prevalence rate (38.5%, 30/78) of carbapenemase producers. Additionally, most isolates harbored MDR genes, including Extended spectrum β-lactamases (ESBLs), AmpC, and quinolone and aminoglycoside resistance genes. Loss of porin genes was observed, and Class 1 integron was detected in 66.7% of the investigated isolates. PFGE and MLST results excluded the occurrence of clonal dissemination among these isolates. CONCLUSIONS: A high prevalence of NDM-1 genes encoding carbapenem resistance determinants was demonstrated among the K. pneumoniae isolates. Importantly, this is the first report of bla(NDM-1) carriage in a K. pneumoniae ST1383 clone in China and of a MDR CRKP isolate co-harboring bla(NDM-1), bla(KPC-2), bla(CTX-M), bla(SHV), acc(6′)-Ib, rmtB, qnrB, and acc(6′)-Ib-cr.
China* ; Clone Cells ; Drug Resistance, Bacterial ; Drug Resistance, Microbial ; Drug Resistance, Multiple* ; Electrophoresis, Gel, Pulsed-Field ; Genes, MDR ; Integrons ; Klebsiella pneumoniae* ; Klebsiella* ; Molecular Epidemiology ; Phenotype ; Plasmids ; Pneumonia ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Prevalence ; Replicon

OBJECTIVETo investigate the HBV quasispecies groups in the patients with chronic HBV infection.

METHODSA set of specific primers was synthesized according to DNA sequence of HBV strain found in China. The whole core promoter (CP) region was amplified by PCR method from the sera of 3 patients with chronic HBV infection, and the PCR products were subcloned into pGEM Teasy vectors. The clones were randomly selected to be sequenced. Sequence comparison of the selected clones was made to find the difference.

RESULTSBy comparison, it was found that each sequence of selected clones was different. The point mutation always occurred in TATA-like boxes, especially from T to C replacement on 184 site. There is a hot region (33.3% 5/15) in basic core promoter where deletion mutation frequently happened.

CONCLUSIONSThere is a hot deletion region near DR I in CP. The replacement at 184 nt (T to C) in the third TATA-like box may influence the expression of pre-C/C protein. The sequencing results suggest that there are HBV quasispecies groups in chronically infected patients.

DNA, Viral ; analysis ; Genes, Viral ; genetics ; Genetic Variation ; Hepatitis B Core Antigens ; genetics ; Hepatitis B virus ; classification ; genetics ; Hepatitis B, Chronic ; virology ; Humans ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; TATA Box ; genetics

OBJECTIVETo identify human single chain Fv antibody (ScFv) against hepatitis B viral surface antigen.

METHODSThe recombinant phages were panned by HBsAg which was coated in a microtiter plate, after five rounds of biopanning, 56 phage clones were identified specific to HBsAg. The specificity of ScFv was evaluated by ELISA and immunohistochemistry, respectively.

RESULTSThe data of HB sAg-ScFv DNA digestion and DNA sequencing showed that the ScFv gene is composed of 750 bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis B surface antigen has a specific combination character with hepatitis B surface antigen of different sources and paraffin-embedded patients tissue specimens, it did not react with normal liver tissue and HCV.

CONCLUSIONSThe application of HBsAg specific ScFv in immunohistochemistry was successfully achieved.

Bacteriophages ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B Antibodies ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B, Chronic ; diagnosis ; Humans ; Peptide Library