Establishment of a stable cell line replicating hepatitis B virus DNA carrying the reverse transcriptase region derived from a clinical isolate / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a stable cell line that can replicate hepatitis B virus (HBV) DNA carrying the reverse transcriptase sequence derived from a clinical isolate.</p><p><b>METHODS</b>Nested PCR was used to amplify the HBV DNA fragment from the serum. The fragment was cloned into a plasmid that can support HBV replication in vitro by fragment substitution reaction (FSR), followed by the cloning of the neomycin expressing fragment downstream from HBV DNA. G418 selection was conducted after the transfection of HepG2 cells with the recombinant DNA. Real-time PCR and enzyme linked immunosorbent assay (ELISA) were used to screen stable cell lines that can replicate HBV DNA, and the replication of HBV DNA by the cell line was confirmed by using Southern blot analysis.</p><p><b>RESULTS</b>Fragment nt55-1654 amplified from the serum DNA was substituted to the plasmid pLL, generating the plasmid p11. The neomycin fragment was cloned into p11, leading to the plasmid p11-neo, and p11-neo was confirmed to be HBV-replication-competent. A stable cell line named 3-10 that can replicate HBV DNA was obtained.</p><p><b>CONCLUSIONS</b>A stable cell line was established that can replicate HBV DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Real-time PCR plus ELISA may help to rapidly screen out stable cell lines replicating HBV DNA.</p>

Optimization and assessment of a reverse hybridization system for the detection of HBV drug-resistant mutations / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a detection method for HBV drug-resistant mutations related to lamivudine, adefovir and entecavir by optimization and assessment of reverse hybridization system.</p><p><b>METHOD</b>26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane. PCR products labeled with digoxigenin were hybridized with corresponding probes. To improve the sensitivity and specificity, 4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin, the energy intensity of UV cross-linking, hybridization and stringency wash conditions. To prove the feasibility, the specificity, sensitivity and accuracy of this system were assessed respectively.</p><p><b>RESULT</b>Sensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenin, energy intensity of UV cross-linking for 1500 x 0.1 mJ/cm², hybridization at 42 degrees C and stringency wash with 0.5 x SSC and 0.1% SDS solution at 44 degrees C for 30 min. In the assessment of system, the majority of probes have high specificity. The quantity of PCR product with a concentration of 10 ng/μl or above can be detected by this method. The concordant rate between reverse hybridization and direct sequencing is 93.9% in the clinical sample test.</p><p><b>CONCLUSION</b>Though the specificity of several probes needs to be improved further, it is a simple, rapid and sensitive method which can detect HBV resistant mutations related to lamivudine, adefovir and entecavir simultaneously. Due to the short distance between 180 and 181, likewise 202 and 204, the sequence of the same probe covers two codon positions, and hybridization will be interfered by each other. To avoid such interference, the possible solution is that probes are designed by arranging and combining various forms of two near codons.</p>

Detection of HBV resistant mutations related to lamivudine, adefovir and entecavir by reverse hybridization technique / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To establish a method for simultaneous detection of HBV resistant mutations associated with three kinds of nucleoside analogues.</p><p><b>METHODS</b>According to 981 HBV complete sequences in GenBank, two pairs of conserved primers labeled with digoxigenin were synthesized to amplify the region of HBV reverse transcriptase. To detect non-synonymous amino acid substitutions associated with lamivudine, adefovir and entecavir, 26 specific oligonucleotide probes covering ten different codon positions, I169T, V173L/G, L180M, A181T/V, T184G, S202I/G, M204V/I, Q215S, N236T and M250V/I/L were synthesized and immobilized on nylon membranes charged positively. The oligonucleotide probes immobilized on nylon membranes were then hybridized with PCR products labeled with digoxigenin to detect three drug-resistant mutations. In order to observe specificity and accuracy of probes, HBV wild-type, resistant reference strains and patients serums were assayed by reverse hybridization technique, respectively.</p><p><b>RESULTS</b>The specific probes of 10 codon positions related to HBV wild-type and resistant reference strains, including I169T, V173L, L180M, A181T, T184G, S202I, M204V, Q215S, N236T, M250V, were distinguished effectively by reverse hybridization method. The results results of 37 samples applicated the method were in accordance with that Of DNA sequencing.</p><p><b>CONCLUSION</b>Reverse hybridization technique can be applied to detect HBV resistant mutations associated with Lamivudine, Adefovir and Entecavir rapidly and accurately.</p>

Elastic intramedullary nail for treatment of extremity fractures in children / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To assess the clinical therapeutic effects of elastic intramedullary nail on extremity fractures in children.</p><p><b>METHODS</b>From June 2005 to March 2008, 40 children with extremity fractures were treated by elastic intramedullary nail, in whom femoral shaft fractures occurred in 26 cases, tibiofibular fractures in 8 cases, radial capitular fractures in 4 cases, ulnoradial fractures in 2 cases. All patients were treated by closed reduction and elastic intramedullary nail fixation.</p><p><b>RESULTS</b>All the fractures gained satisfactory reduction and healing. The average duration needed for fracture healing was 1-2 months. Postoperative follow-up confirmed a sound functional recovery.</p><p><b>CONCLUSIONS</b>The elastic intramedullary nail is a minimally invasive and effective surgical approach for treatment of extremity fractures in children. It allows early functional exercises after operation and secures a satisfactory bone union and functional recovery.</p>

The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV / 中国病毒学

To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1) encoded by Epstein-Barr virus(pshLMP1), and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells, we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis, RT-PCR and western blot. pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method. Furthermore, the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors. According to our research, we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells, and also provides a novel application of RNA interference technology against-EBV.

Regulation of the hepatitis B virus X promoter activity by a novel negative regulatory element / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To learn the effect of hepatitis B virus (HBV) sequence nt250-453 on the HBV X promoter.</p><p><b>METHODS</b>A plasmid pNRE-XP which contains the NRE and the HBV X promoter was constructed to co-transfect HepG2 cell line with plasmid RL-TK. The firefly luciferase activity and mRNA expression of the firefly luciferase gene were both detected. Then, nt250-453 of HBV was removed from LJ196, which contained HBV full genes. The mutated plasmid LJ196 and plasmid LJ96 which provided core protein and the viral DNA polymerase were used to co-transfect HepG2 cell line. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the X gene mRNA level.</p><p><b>RESULTS</b>The activity of firefly luciferase and the expression of firefly luciferase gene mRNA were both down-regulated in the presence of the NRE, while the HBV X gene mRNA expression increased as it was removed from the HBV genes.</p><p><b>CONCLUSION</b>This study demonstrates that nt250-453 of HBV acts as a novel negative regulatory element which could suppress the HBV X promoter activity.</p>

Development of a Reverse Dot Blot System for Rapid Detection of HBV Genotypes / 中国生物工程杂志

To develop a reverse dot blot assay for rapid detection of HBV genotypes.Specific oligonucleotides probes were desighed and immobilized on nylon membranes.The DNA sample to be tested was PCR-amplified with DIG labeling primers and then hybridized with the immobilized probes.This procedure for detecting HBV genotypes was simple,rapid and specificity.30 specimens in Chongqing area were collected and detected by this method,and results were evaluated using direct sequencing.Results showed that: This new method was applicable to precise detection HBV genotypes for specimen with copies up to 103,and the HBV genotyping results showed that genotype B was the predominant genotype in Chongqing area.