Animals ; Disease Models, Animal ; Hepatitis B ; physiopathology ; Hepatitis B virus ; physiology

To establish a cell model in vitro that stable expressing HBV by integrating HBA1.3 DNA into cell chromosome. The HBV1.3 full-length DNA was obtained by digested pGEM-HBV1.3 plasmid with HindIII and then was linked with PU-21 vector digested by HindIII. This was resulted in generation of a recombined plasmid named PU21-HBV plasmid. The recombined plasmid was introduced into HepG2 cells by electroporation. The transfected cells were screened with G418. The insertion and expression of HBV were identified by X-gal staining, RT-PCR and Southern blot. The result of PU21-HBV plasmid sequence demonstrated that HBV1.3 DNA was linked correctly with PU-21 vector. A series of positive cell colonies were obtained with G418 screening followed transfecting PU21-HBV plasmid into HepG2 cells. The results of Southern blot and RT-PCR exhibit that HBV1.3 DNA had successfully integrated into the chromosomes of HepG2 cells and had functional HBV gene transcription. HBV1.3 DNA was inserted into HepG2 genome and could stable transcript HBV RNA. The stable HBV expression cell line was constructed successfully. There are LoxP sites in the trapping vector PU21. With the Cre enzyme, interesting genes could be excganged into the LoxP sites. Therefore, double stable expression of interesting gene and HBV cell lines could be generated. The cell lines will be useful for further research some target gene function on replication of HBV.

Objective To demonstrate the carbonic anhydrase Ⅲ (CAⅢ) for 25 000 protein decreased in skeletal muscle of myasthenia gravis (MG). Methods The protein molecular properties responsible to antibodies against 25 000 protein and CAⅢ were analyzed by a combination method of two-dimensional electrophoresis and immuno-Western blot. Competitive binding reactions of the antibodies to the purified 25 000 protein and muscular homogenate were observed by using immuno-Dot blot and immuno-Western blot, respectively. The expression of CAⅢ from normal and MG muscles was detected by immuno-Western blot. Results Combination analysis of two-dimensional electrophoresis and immuno-Western blot showed that the protein of immunological responsible to antibodies against 25 000 protein and CAⅢ had an identical molecular mass and isoelectric point. Competitive binding reactions proved that 25 000 protein and CAⅢ were the same substance, either by immuno-Dot blot or by immuno-Western blot. In addition, a much similar result was obtained when the levels of 25 000 protein from normal and MG muscles were detected by antibodies against 25 000 protein and (CAⅢ) by immuno-Western blot. Conclusion 25 000 protein decreased in the MG skeletal muscle was proved to be just a known protein CAⅢ, which made a basis for further exploring the relationship of CAⅢ deficiency and MG pathogenesis.

To counteract the immune system in parasitic hosts, some viruses encode proteins to suppress the RNA interference (RNAi) effect. In this report, we established two RNAi systems to be easily observed with strong and obvious effect. The function of the P19 of tomato bushy stunt virus, which suppresses RNAi in mammal cells, was then studied using these two systems. Short hairpin RNAs targeting green fluorescence protein (pshRNA-GFP) and firefly luciferase (pshRNA-luc) were designed and inserted into a eukaryotic transcriptional vector pTZU6+1, respectively. The shRNA expressing vectors were co-transfected with plasmids containing the target gene with or without P19. The GFP expression level was assayed by fluorescence microscopy, Western blotting and RT-PCR. The luciferase expression level was analyzed by the dual-luciferase assay system. pshRNA designed in this study down-regulated the target gene specifically and efficiently, with a decrease of expression of both genes of about 70%, respectively. When P19 was introduced into the RNAi systems, the expression of both GFP and the luciferase were mostly recovered compared with the control groups. The RNAi systems of GFP and luciferase were constructed successfully, demonstrating that P19 of tomato bushy stunt virus has the ability to counteract the RNAi effect induced by shRNA in mammal cells.

OBJECTIVETo establish a set of suitable and reliable methods for HBV genotyping and to study the distribution of HBV genotypes.

METHODSType-specific nucleotides were searched through alignment of S genes (more than 1000 sequences) listed in GenBank. Then, type-specific primers were designed and type-specific primer PCR was used to genotype the 238 HBV strains. S genes of the untyped strains were further amplified and sequenced to find out their genotypes with type-specific nucleotide analysis.

RESULTSAll the 238 HBV strains were genotyped. 159 (66.8%) cases were genotype B, 69 (28.9%) were genotype C, 6 (2.5%) were mixtures of genotypes B and C and 4 (1.6%) were mixtures of genotypes B and D. No genotypes of A, E, F, G, and H were found.

CONCLUSIONGenotypes B and C are the most common types for HBV strains. Mixtures of genotypes B and C or genotypes B and D coinfection rarely existed. There is no relationship between the gender of the patients and HBV genotypes (X2 = 0.794, P more than 0.05).

DNA Primers ; DNA, Viral ; blood ; genetics ; Female ; Genotype ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; virology ; Humans ; Male ; Nucleotides ; genetics ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA

OBJECTIVETo compare the sensitivity of different hepatocellular carcinoma (HCC) cell lines (HepG2, QGY7701, HepG2.2.15) and the normal liver cell line L02 to 5-aza-dC, an DNA methyltransferase inhibitor, and to explore the relationship between global DNA methylation level and the sensitivity to 5-aza-dC.

METHODSHepG2, QGY7701, HepG2.2.15 and L02 cells were treated with 5-aza-dC at different concentration, cell proliferation was measured by MTT method, cell apoptosis was detected by measuring caspase 3 activity and cellular DNA fragmentation ELISA.

RESULTSCompared to HepG2 and QGY7701 cells, HepG2.2.15 were less sensitive to the treatment of 5-aza-dC; the normal liver cell line L02 was less sensitive to 5-aza-dC than the HCC cell lines.

CONCLUSIONSThe sensitivity to 5-aza-dC of HCC cell lines and normal liver cells is not correlated with the global DNA methylation level.

Azacitidine ; analogs & derivatives ; pharmacology ; Carcinoma, Hepatocellular ; metabolism ; Caspase 3 ; metabolism ; DNA Methylation ; drug effects ; DNA Modification Methylases ; antagonists & inhibitors ; Hep G2 Cells ; Humans

Animals ; Behavior, Animal ; drug effects ; Depressive Disorder ; drug therapy ; psychology ; Eleutherococcus ; Male ; Plant Extracts ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Stress, Psychological

Gene Silencing ; Genetic Therapy ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; therapy ; virology ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics

OBJECTIVETo clone and analyze duck hepatitis B virus genome from Chongqing brown duck.

METHODSDuck hepatitis B virus (DHBV) DNA extracted from a Chongqing brown duck was amplified by PCR and cloned into PGEM-T vector using T-A clone method. The sequence of this DHBV genome was analyzed with some softwares after identified.

RESULTSThe duck hepatitis B virus genome from Chongqing brown duck (DHBVcq), which was 3 024 nucleotides long, contained three ORFs whose onset and end nucleotides were in accord with those of HPUGA, encoding P, PreC/C and PreS/S protein respectively. Comparison of this strain with other DHBV reported in GenBank showed that the homology of DHBVcq and M32990 got the highest score of 94.9% at nucleotide level, while DHBVcq and DHBVCG got the least (89.8%). Most of the conserved regulation nucleotides and amino acids sequence found in other DHBV were also identified in DHBVcq. The epsilon region of DHBVcq, which was important for encapsidation of pgRNA and synthesis of minus-strand DNA, differed from that of most other DHBV strains, forming a stem-loop conformation with a three- nucleotides upper stem rather than a common nine-nucleotides one in free status.

CONCLUSIONThe successful clone and analysis of DHBVcq provide further studies with helpful information.

Animals ; Cloning, Molecular ; DNA, Viral ; chemistry ; genetics ; Ducks ; Hepatitis B Virus, Duck ; classification ; genetics ; Hepatitis Virus, Duck ; genetics ; Open Reading Frames ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Sequence Homology