Comparative analysis of drug resistant mutations in the reverse transcriptase domain of hepatitis B virus covalently closed circular DNA and the viral relax circle DNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a method for detection of reverse transcriptase region of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to compare the pattern and frequency of drug-resistant mutations in the region between intrahepatic HBV cccDNA and serum HBV relax circle DNA (rcDNA).</p><p><b>METHODS</b>HBV DNA were extracted from liver biopsy tissues of 20 patients with chronic hepatitis B. The RT region of HBV cccDNA was amplified by rolling circle amplification (RCA) followed by polymerase chain reaction (PCR) mediated by a pair of primers spanning across the gap region of HBV genome. The RT region of serum HBV rcDNA from the same patient was amplified by nested-PCR. The PCR products were directly sequenced and analyzed by Vector NTI Suite 8.0 and chromaslite 201 software. x2 test was used for statistical significance analysis of drug-resistant mutation occurrences between the HBV cccDNA and rcDNA.</p><p><b>RESULTS</b>The RT regions of HBV cccDNA were successfully amplified from liver tissues of all enrolled patients using the RCA plus PCR assay. Simultaneously, HBV the RT regions of rcDNA were amplified from these patients serum samples. Sequence analysis showed that the drug-resistant mutations were significantly more frequently detected in HBV rcDNA (40%) than in HBV cccDNA (10%) (P<0.05). Different mutational patterns were observed between the HBV cccDNA and rcDNA in a few cases.</p><p><b>CONCLUSION</b>The RCA in combination with PCR is a practical method for the detection of drug-resistant mutation in the RT region of HBV cccDNA. Drug-resistant mutational patterns could be discrepant between HBV cccDNA and rcDNA.</p>

Epitope screening of influenza A (H3N2) by using phage display library / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To screen the influenza A (H3N2) mimotopes by using phage display library.</p><p><b>METHODS</b>Using influenza A (H3N2) monoclonal antibody as selective molecule, a 7 mer phage peptide library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing.</p><p><b>RESULTS</b>21 positive clones were chosen for DNA sequencing. From the experiment and sequencing comparison results, one epitope was comfirmed as mimotope of influenza A (H3N2).</p><p><b>CONCLUSION</b>Influenza A (H3N2) mimotope was obtained by phage peptide library screening. The result provide a new approach for new Influenza virals vaccine development.</p>

Analysis and recombinant construction of HBV the reverse transcriptase gene with drug-resistant mutations from 40 patients with chronic hepatitis B / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze genetic mutation associated with drug resistance in the reverse transcriptase (RT) domain of HBV from 40 patients with chronic hepatitis B, and to construct mutant RT gene recombinant vectors for drug-resistant phenotypic analysis.</p><p><b>METHODS</b>HBV DNA was extracted from sera of the 40 patients receiving anti-HBV nucleot (5) ide analogue. The complete RT domain-encoding gene was amplified by nested PCR, and then cloned into pGEM-T-easy vector. Three to Five clones were randomly selected for DNA sequencing. Data were analyzed by UNASTAR software. The pTriEx-HBV (C) 1.1 expression vectors were constructed by replacing the 1250-hp Xho I/Nco I fragments containing complete RT domain from individual patients samples.</p><p><b>RESULTS</b>All samples were detected with drug-resistant mutations associated with lamivudine, adefovir, and entacavir singly or in combination. Ninety-six mutant RT genes were cloned into pGEM-T-easy vector, from which 40 major mutant RT genes were replaced into pTriEx-HBV (C) 1.1 expression vectors. The construction was confinned to be successful by verifying mutation existence using DNA sequencing, and detectable HBsAg and HBeAg in the cell supernatant after transfecting recombinant expression vectors into Huh7 cells.</p><p><b>CONCLUSION</b>The analysis of drug-resistant mutation and the construction of mutant-recombinant expression vectors were successfully implemented using the samples frum clinical patients. The work lays a foundation for drug-resistant phenotypic analysis of HBV mutants.</p>

Quantitative detection of hepatitis B virus covalently closed circular DNA in sera of chronic hepatitis B patients with a newly established assay / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To quantitatively detect hepatitis B virus covalently closed circular DNA (HBV cccDNA) in sera of chronic hepatitis B patients with a newly established assay.</p><p><b>METHODS</b>Primers and probe were designed in highly conservative region of HBV DNA. DNA was extracted from 175 sera samples of chronic hepatitis B patients, and was treated with plasmid-Safe-ATP-dependent Dnase(PSAD) to eliminate the relaxed circular DNA (rcDNA). The products were amplified by real-time PCR with primers spanning.</p><p><b>RESULTS</b>The detection rate of serum HBV cccDNA was found to correlate directly with serum HBV DNA loading. HBeAg positive chronic hepatitis B patients had higher serum HBV cccDNA levels than HBeAg negative chronic hepatitis B patients.</p><p><b>CONCLUSION</b>The method is good because of the high specificity. It can be used for detection of HBV cccDNA. DNA;</p>

Multiple-site analysis of HBV drug-resistant mutations in 340 patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To analyze HBV drug-resistant mutations against nucleos(t)ide analogues at 12 reported sites in 340 patients with chronic hepatitis B.</p><p><b>METHODS</b>Serum HBV DNA was extracted and a nested PCR assay was employed for the reverse transcriptase (RT) gene amplification. Direct sequencing of PCR product was performed. The significance of detected mutations was analyzed in view of clinical data of the patients.</p><p><b>RESULTS</b>Drug-resistant mutations were detected in 68 patients taking lamivudine (LAM), 10 taking adefovir (ADV), 8 taking entecavir, and 1 taking telbivudine (LdT). M204V and M204I were the most common LAM-resistant mutations. The former usually emerged with L180M while the latter often emerged alone. N236T +/- A181 substitution was the most frequently seen ADV-resistant mutation. ETV-resistant mutations occurred on the basis of LAM-resistant mutations and T184 change was the most common form. LdT-resistance was observed as M204I. Interestingly, these drug-resistant mutations were detected in a few patients who had not been treated with nucleos(t)ide analogues.</p><p><b>CONCLUSION</b>Detection of HBV drug-resistant mutations at multiple sites of the viral RT gene is valuable for discovering and verifying drug resistance and thus is very helpful in planning anti-HBV therapy.</p>

Screening and identification of proteins interacting with HCV NS4A via yeast double hybridization in leukocytes and gene cloning of the interacting protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To screen proteins interacting with HCV NS4A protein in leukocytes by yeast-double hybridization.</p><p><b>METHODS</b>The bait plasmid pGBKT7-NS4A was transformed into yeast AH109 was transformed, and the expressing of the fusion protein was identified by SDS-page. The transformed yeast was mated with yeast Y187 containing leukocytes cDNA library plasmid in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, analysis was conducted by bioinformatics. And, the gene encoding the interesting protein was cloned, and back-cross was performed.</p><p><b>RESULTS</b>Forty-five colonies were sequenced, among them, 29 colonies were human calcium modulating cyclophilin ligand (CAML). The gene encoding CAML was cloned, and the interaction between NS4A and CAML was ensured.</p><p><b>CONCLUSION</b>Seven kinds of proteins interacting with NS4A in leukocytes were successfully screened and the results brought some new clues for studying the pathogenesis of HCV.</p>

Identification and analysis of a mouse gene homologous to human hepatitis B virus pre-S1 protein-binding protein using the bioinformatics method / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To clone and identify the mouse gene homologous to human hepatitis B virus (HBV) pre-S1 protein-binding protein (PS1BP).</p><p><b>METHODS</b>The human PS1BP cDNA sequence was used as the reference sequence to search homologous mouse cDNA sequence from GenBank established by National Center for Biotechnology (NCBI), National Institute of Health (NIH), for its homologous cDNA sequences of mouse by BLASTn tool. The characteristics of mouse PS1BP protein primary structure were predicted by online software. Finally the genomic DNA structure of mouse PS1BP was deduced and compared.</p><p><b>RESULTS</b>The mouse PS1BP was identified and consisted of 1455 nt, coding a protein of 484 aa. The identity of human and mouse PS1BP protein is 84.92% (411/484). The genomic DNA of mouse PS1BP consisted of 3 exons and 2 introns.</p><p><b>CONCLUSION</b>The identification and characterization of mouse PS1BP cDNA and genomic DNA pave a way for further study of their structures and functions.</p>

Screening of binding proteins to interferon-alpha promoter DNA by phage display technique / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the interferon alpha regulation mechanisms by screening binding proteins of interferon alpha promoter by phage display.</p><p><b>METHODS</b>PCR product of interferon-alpha promoter was incubated with a phage display cDNA library that expressed a library of human liver proteins on the surface of bacteriophage T7. Unbound phages were washed off and the phages bound to the interferon alpha promoter were amplified. Positive plaques were amplified by PCR and cloned into a pGEM-Teasy vector in order to perform DNA sequence analysis and subsequent computer blasting analysis.</p><p><b>RESULTS</b>Positive phage-displayed proteins binding with interferon alpha promoter were enriched after five rounds of biopanning. We found that the following proteins were relevant to interferon alpha: mitochondrial ribosomal protein, chromosome clone, fibrinogen A alpha polypeptide, enoyl coenzyme A hydratase short chain, eukaryotic translation elongation factor 1 alpha, PI-3-kinase-related kinase SMG-1-like, xeroderma pigmentosum C group, and Homo sapien activity-dependent neuroprotector (ADNP).</p><p><b>CONCLUSION</b>Many proteins with different functions could bind with interferon alpha promoter.</p>

Synergetic transactivating effect of HCV core and HBV X proteins on SV40 early promoter/enhancer / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate the synergetic transactivating effects of HCV core and HBV X proteins.</p><p><b>METHODS</b>HCV core and HBV X protein-expressing plasmids were constructed with the vector pcDNA3.1(-). The plasmids were transfected into HepG2 cells and cotransfected Hep2 cells with reporter plasmid Psv-lacZ by lipofectamine plus reagents. The virus proteins produced in transient expression system were detected at the transcription and translation levels. The activity of b-galactosidase was detected, which reflected the transactivating function of the proteins.</p><p><b>RESULTS</b>The expression of plasmids were detected in soluble protein cell extracts of transiently transfected HepG2 cells. HCV core protein activated the b-galactosidase expression at a value 4.9 times higher than the control, while HBV X protein activated at a value 3.5 times. It arrived at 9 times transfected with the plasmids simultaneously. The activating effect increased in relation to the amount of plasmids.</p><p><b>CONCLUSIONS</b>The results suggested that the two kinds of virus proteins have transactivating effect on SV40 early promoter/enhancer, and they acted synergistically. These contribute to explain the mechanisms of liver injury or tumorigenesis induced by HCV or/and HBV infection.</p>

Cloning and expression of human single-chain Fv antibody against amyloid beta peptide involved in Alzheimer's disease / 中国医学科学院学报

<p><b>OBJECTIVE</b>To screen out the specific antibody clones against amyloid beta peptide 40, and clone the antibody gene and express it in a bacterial system, so as to provide a solid basis for novel diagnostic and therapeutic methods for Alzheimer's Disease.</p><p><b>METHODS</b>beta amyloid peptide 40 was bound on the solid surface of Nunc plates as antigen to screen the binding clones from a phage-display human single-chain Fv antibody library. After five rounds of bio-panning, the host E. coli TG1 was infected with eluted filamentous phage from the last turn of selection. 55 well-separated colonies were picked randomly from the plates and the specific positive clones were identified by ELISA test. The single-chain Fv antibody gene was sequenced and their amino acids sequence was deduced. The scFv antibody gene was sub-cloned into a protokayotic expression vector pGEX-6P-1 and transformed into bacteria strain BL21 to express the glutathione-S-transferase (GST) fusion single-chain antibody.</p><p><b>RESULTS</b>ELISA test showed that 33 of the 55 clones could bind amyloid beta peptide 40 and 10 of the 33 clones could be inhibited by amyloid beta peptide 40 itself to below 50% of its original binding activities. Five of the 10 clones could also be inhibited by amyloid beta peptide 1-16 to the same level, which meant that the binding epitope of the antibody from the 5 clones was between first to sixteenth amino acids at amino-end of amyloid beta peptide 40. DNA sequencing data demonstrated that the gene of the single-chain antibody specifically against amyloid beta peptide 40 was consisted of 768 bp and the deduced amino acids sequence confirmed its typical antibody structure. The complement determinant regions and framework regions were discriminated empirically. After cloning the antibody gene into a protokayotic system, the GST fusion antibody was expressed as the expected size.</p><p><b>CONCLUSIONS</b>After five rounds of bio-panning and subsequently serial ELISA testing, the specific antibody clones against amyloid beta peptide 40 were screened out successfully. The antibody gene DNA sequence and amino acids sequence were analyzed and confirmed. The fusion antibody was expressed as expected in the bacterial system.</p>

Epitope mapping of hepatitis C virus non-structure protein 5 from a 7 peptide phage library by using immobilized specific monoclonal antibody / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To screen HCV NS5 mimotopes by using monoclonal antibody and phage peptide library.</p><p><b>METHODS</b>By using HCV NS5 monoclonal antibody as selective molecule, a 7 peptide phage library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing.</p><p><b>RESULTS</b>Twelve positive clones were chosen for DNA sequencing. From the experiment and sequencing comparison results, one epitope was confirmed as the mimotope of HCV NS5.</p><p><b>CONCLUSIONS</b>HCV mimotope is obtained by phage peptide library screening. The result provides a new approach for HCV therapy and vaccine development.</p>

Study on the interaction between hepatitis virus C nonstructural protein 4A and calcium modulating cyclophipin ligand by in vivo coimmunoprecipitation / 中华传染病杂志

Objective To prove the interaction between hepatitis virus C(HCV)nonstruetural protein 4A(HCV NS4A)and calcium modulating cyclophilin tigand(CAML)with yeast-two hybrid- ization and coimmunoprecipitation.Methods The gene encoding CAML was cloned,and subcloned into the yeast expression vector pGADT7 and eucell expression vector pcDNA3.1/His-A.The back- cross test between HCV NS4A and CAML was performed in yeast cells.After that,the pCMV-Myc/ NS4A plasmid and pcDNA3.1/His-A-CAML plasmid were co transfected into 293 cells and,then, coimmunoprecipitation and Western blot were performed.Results The gene encoding CAML was cloned sucessfully,and then the gene was subcloned into yeast expression vectors,pGADT7.After the interaction between NS4A and CAML was ensured in yeast cells,the eukaryotic expression vec- tors of NS4A and CAML were constructed and their interaction was ensured again by Co-immunopre- cipitation.Conclusions The interaction between HCV NS4A and CAML is proved.CAML is one of the proteins involved in Ca~(2+)signaling,which suggests that the interaction of HCV NS4A and CAML may be a new clue of the chronic mechanism of HCV infection.Future studies will be required to de- fine the physiologic significance of this interaction.