Biomedical subjects

Ni Tang

Publications and source records attributed to Ni Tang.

23 records · Page 2Linked to original sources

PubMed2003

[Sequence analysis of cloned duck hepatitis B virus genome from a Chongqing brown duck].

OBJECTIVE: To clone and analyze duck hepatitis B virus genome from Chongqing brown duck. METHODS: Duck 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. RESULTS: The 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. CONCLUSION: The successful clone and analysis of DHBVcq provide further studies with helpful information.

Animals↗

PubMed2003

[Construction of recombinant eukaryotic expression plasmid containing 1.3-fold-overlength genome of HBV and its expression in HepG2 cells].

OBJECTIVES: To transfer 1.3-fold-overlength genome of HBV expression plasmid into HepG2 cells, and observe the dynamic changes of viral replication as well as expression in the transfected cells. METHODS: 4.1 kb fragment of HBV genome, derived from pGEM-HBV, was cloned into Hind III site of the eukaryotic expression vector pCDNA3.1 to construct the recombinant plasmid pHBV. Then HepG2 hepatoma, cells were transfected with pHBV, using Lipofectamine2000 transfection reagent. After 24, 48, 72 hours, the levels of HBsAg and HBeAg in the supernatant of HepG2 cells were determined with Abbott MEIA Kit. Intracellular viral DNA and RNA were analyzed by Southern and northern blot hybridization. In addition, viral-specific proteins (HBsAg and HBcAg) were assayed by immunofluorescence staining. RESULTS: The expression vector pCDNA3.1 was constructed successfully. The levels of HBsAg were 5.36+-0.25, 13.42+-1.24, 7.52+-0.43, and the values of HBeAg were 9.16+-0.32, 22.75+-1.49, 15.96+-1.03 after 24, 48, 72 hours, respectively. All expected HBV replicative intermediates and specific transcripts were verified by Southern and northern blot analysis. The HBsAg-positive cells peaked after 24 hours, and then dropped slowly. HBsAg positive staining scattered in the cytoplasm, whereas HBcAg lied maily in the cytoplasm apart from nuclears. CONCLUSIONS: This recombinant plasmid, which initiates viral replication efficiently in infected cells, is expected as a novel tool for investigating HBV replication in vitro.

Carcinoma, Hepatocellular↗

PubMed2003

[A new method for amplification and sequence analysis of the full-length of HBV genome].

OBJECTIVES: To develop a new method for amplifying and sequencing the full-length of HBV genome. METHODS: A pair of primers located at the nick region of HBV molecule and a thermostable polymerase with high fidelity and sensitivity were used. After cloning the PCR products into a plasmid, the sequences of HBV genome were analyzed. RESULTS: The full-length of HBV genome were acquired using this method. The sensitivity and fidelity of the new method were also analyzed. The least quantity of initial templates was 10(2) and the artificial mutation rate was 1.2 bp/kb. CONCLUSION: This method can be used in amplification and sequence analysis of the full-length of HBV genome on a large scale.

DNA Primers↗

PubMed2003

[Inhibition of survivin expression in liver cancer cells by shRNA].

OBJECTIVE: To construct the plasmid containing short hairpin RNA (shRNA) of survivin in order to suppress the expression of survivin gene in HepG2 and SMMC-7721. METHODS: Two 20 to 21 bp reverse repeated motifs of survivin target sequence with 4 bp or 8 bp spacer were synthesized respectively and inserted into plasmid pTZU6+1 to generate the plasmid pshRNA-survivin1 and pshRNA-survivin2; plasmid pEGFP-C1-survivin and pshRNA-survivin1 or pshRNA-survivin2 plasmid were cotransfected into liver cancer cell HepG2 and SMMC-7721 to detect effect of GFP expression respectively and analyze the inhibition of survivin gene. RESULTS: The recombinant plasmid pshRNA-survivin1 and pshRNA-survivin2 were successfully constructed. The recombinant plasmids suppress the survivin expression by 80% in HepG2 and SMMC-7721. CONCLUSION: The result showed that the short hairpin RNA of survivin can efficiently suppress it's expression in HepG2 and SMMC-7721.

Gene Expression Regulation, Neoplastic↗

PubMed2002

[Replication and transfection of hepatitis B virus DNA into primary duck hepatocytes].

OBJECTIVE: By studying the possibility of obtaining expression of human hepatitis B virus (HBV) genes and production in normal liver cells from heterologous species like normal primary duck hepatocytes (PDH), to investigate the species-specificity of HBV infection and replication. METHODS: Two days after transfecting the complete HBV genome into PDH by electroporation (transfected group), HBsAg and HBeAg in the supernatants and lysates of PDH were measured by the IMX system. Meanwhile, replication of HBV in PDH was analyzed by Southern blotting and dot blotting procedures. PDH was electroporated as control. RESULTS: HBsAg in the lysate of transfected group was 9.10 (P/N values, positive?2.1), HBeAg was 1.0 (negative?2.1), both were negative in the supernatants of transfected group. dot blotting revealed that transfected group was strongly positive, whereas the control group was negative. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (RC), covalently closed circular (CCC) and single-stranded (SS) HBV DNA replicative intermediates in the transfected group, and there was no integrated HBV DNA in the cellular genome. Control groups were negative. CONCLUSIONS: Replication of HBV can occur in hepatocytes from nonmammalian species, which strongly supports the idea that replication of HBV has no critical species-specificity, and yet it depends on the endoenvironment of hepatocyte.

Animals↗