Objective To construct plasmid pVR1012 M as nuclei acid vaccine for hepatitis B,was constructed to immunize mice with or without plasmid pcDNA 3.1 - IL 18 to identify the effect of Interleukin 18(IL 18). Methods Polymerase chain reaction method was used to amplify the PreS2 and S region of HBV and reconstruct plasmid pVR1012 M as nuclei acid vaccine. Plasmid pcDNA 3.1 - IL 18 was used as a co stimulator. Twenty five Balb/c mice were divided into 3 groups, group 1 immunized with 100 ?g plasmid pVR1012 group 2 pVR1012 M,Group 3,pVR1012 M with pcDNA 3.1 - IL 18, respectively, every 2 weeks for 3 times. Anti HBs were detected in serum 2 weeks after each injection. Lactated ehydrogenase (LDH) cytotoxicity assay was done to analyze the cytotoxic T lymphocytes funciton. Results The positive rate and the antibody titer of serum from mice injected pVR1012 M increasing gradually with the increasing frequency of inoculation, while those from mice injected pVR1012 M and pcDNA 3.1 - IL 18(joint group) were lower than those injected with pVR1012 M alone (Difference after 3rd inoculation was significant, 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

BACKGROUNDTo search a novel sensitive, specific and lower cost method applicable for quantitative analysis of the hepatitis B virus DNA extensively.

METHODSQuantitative analysis of the DNA from 100 sera by real-time PCR with Sybr green 1. The results of Sybr's assay were compared with the results obtained with Taqman's fluorescent quantitative assay.

RESULTSTaqman real-time PCR could help evaluate the level of virus reliably. The results of Sybr's assay were in agreement with the Taqman's assay, but detection rate was lower.

CONCLUSIONSSybr green 1 real-time PCR appeared to be convenient and cheap, but detection rate was lower.

DNA, Viral ; blood ; Fluorescent Dyes ; Hepatitis B virus ; genetics ; Humans ; Organic Chemicals ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

BACKGROUNDTo construct recombinant vector expressing antisense RNA to CCR5 in eukaryotic cells and obtain recombinant pseudovirus, which will be used to block HIV-1 infection.

METHODSThe DNA fragment targeted against the initional part of CCR 5 mRNA translation was amplified by using RT-PCR from peripheral blood mononuclear cells (PBMCs) and cloned into retroviral vector pLXSN, then transfected into packaging cell (PA317) with lipofectAMINE. After 2-3 weeks selecting with G418, the pseudovirion in the survival cell's supernatant was detected with RT-PCR (FQ),then was used to infect NIH/3T3 cell.

RESULTSThe psuedovirion packed from expression vector of sense/antisense RNA to CCR5 had infected NIH/3T3 cell successfully. The vector had incorporated into its genome and transcripted into RNA.

CONCLUSIONSThe gene fragment of antisense RNA to CCR5 could be obtained from PBMCs and transfected into eukaryotic cell with retroviral vector. The results made a great foundation for studying its inhibiting effect on HIV-1 infection.

3T3 Cells ; Animals ; Eukaryotic Cells ; metabolism ; Gene Expression ; Genetic Vectors ; Mice ; Plasmids ; genetics ; RNA, Antisense ; genetics ; Receptors, CCR5 ; genetics ; Transfection

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

BACKGROUNDKnowledge on Hepatitis B surface antigen (HBsAg) kinetics in chronic hepatitis B (CHB) patients with long-term adefovir dipivoxil (ADV) treatment is limited. The aims of this study were to investigate HBsAg kinetics in patients with chronic hepatitis B virus (HBV) infection treated with long-term ADV and to evaluate different characteristics between patients with and without HBsAg loss.

METHODSWe retrospectively evaluated HBsAg kinetics in 24 Chinese patients with chronic HBV infection who achieved continuous virologic suppression during ADV therapy. HBV genotype was determined at baseline. Liver biochemistry, hepatitis B e antigen status, serum HBV DNA, and HBsAg levels were measured at baseline, 6 months, and once every year thereafter.

RESULTSOf these 24 patients, 3, 1, and 20 patients were followed up for 3, 5, and 6 years, respectively. Baseline serum HBsAg level had a moderate correlation with baseline HBV DNA level (r = 0.52, P = 0.01). The median rate of HBsAg reduction during the therapy period was 0.08 lg IU × ml(-1) × y(-1). Baseline serum HBsAg level was significantly higher than other time points (P ranges from 0.046 to 0.002). The HBsAg reduction rate during the first year was similar to that in other years (P > 0.05). The HBsAg reduction rate during the first year in patients with eventual HBsAg loss was significantly faster than that in patients without HBsAg loss (P = 0.005).

CONCLUSIONSSerum HBsAg levels in Chinese CHB patients receiving long-term ADV demonstrated a gradual reduction. Patients with eventual HBsAg loss had a significantly faster HBsAg reduction rate during the first year than those without HBsAg loss.

Adenine ; analogs & derivatives ; therapeutic use ; Adult ; Antiviral Agents ; therapeutic use ; Female ; Hepatitis B Surface Antigens ; blood ; Hepatitis B, Chronic ; blood ; drug therapy ; Humans ; Male ; Middle Aged ; Organophosphonates ; therapeutic use ; Retrospective Studies