Objective:To construct a DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the anti-HBV siRNA genes, and to express this DNA vaccine in HepG2 cells. Methods:The HBV multi-epitope antigen gene was designed and synthesized before it was fused with enhanced green fluorescent protein(EGFP) gene, and cloned into the multi-clone site(MCS) of the eukaryotic expression vector pVAX1. The expressinig units of hIL-12 and siRNA were cloned into the BspH I and Mlu I site of pVAX1 respectively. Then the recombinant plasmid pVAX1-siHBV-HB-EGFP-hIL12 was transiently transfected HepG2 cells. The expression of HBV compound multi-epitope gene was observed through EGFP report gene. The expression of hIL-12 was analyzed by ELISA and the effects of anti-HBV siRNA was confirmed with rtPCR . Results: The analysis of enzyme digestion and sequencing both demonstrated that the trible-expressing HBV DNA vaccine has been constructed successfully. The green fluorescent image was detected in the transfected cells which could confirm the expression of the multi-epitope antigen gene. The amount of hIL-12 secretion was 1289pg/ml in supernatant at 48h after transfection and 1712pg/ml at 72h after transfection. The mRNA amount of HBV S gene, which was the siRNA target, had been obviously knockdown. Conclusion: The DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the siRNA genes was constructed and transiently expressed in HepG2 cells, and siRNA had shown us a good anti-HBV effect. It laid a foundation of further study on anti-HBV effect of the new DNA vaccine.

Objective To identify and rename one strain stored in National Center for Medicine Culture Collections ( CMCC) and used for tracheitis vaccine production. Methods The test strain CMCC (B)29108 and the type strain DSM30007T were cultured on NA medium. Characteristics in morphology, physiology, biochemistry and fatty acid profile were compared between the two strains. Phylogenetic analysis was based on 16S rRNA and rpoB gene sequences, together with the DNA-DNA hybridization assay. Results A Comparative analysis of a partial sequence of the 16S rRNA gene sequence revealed that the CMCC( B) 29108 strain was closed to the Acinetobacter species and showed the highest similarity with the type strain Acinetobacter baumannii DSM30007T. Moreover, the CMCC(B)29108 strain was highly similar to type strain DSM30007T in morphology, physiology, biochemistry and fatty acid profile. On the phylogenetic tree based on 16S rRNA and rpoB gene of all Acinetobacter members, the CMCC(B)29108 strain steadily clustered into one independent branch only with the DSM30007 T strain with a DNA-DNA hybridization value of 100%. Conclusion The CMCC(B)29108 strain that is one of the strains used for the production of tracheitis vac-cine should be assigned to the species of Acinetobacter baumannii based on its phenotypic, phylogenetic and chemotaxonomic characteristics.

Objective To investigate the value of Habib 4X in hepatic resection. Methods The clinical outcome of 21 patients with liver disease who received liver resection at the Tianjin Third Central Hospital from November 2009 to April 2010 were retrospectively evaluated. All the operations were carried out by using Habib 4X. Results All patients received hepatectomy, including right hepatectomy in three patients, left hepatectomy in one patient, multiple segmentectomy in nine patients, single segmentectomy in seven patients and partial liver resection in one patient. All tumors were reseeted completely. The mean operation time was (50±25) minutes and the mean blood loss was(129±117)ml. No patient was transferred to ICU. Three patients were complicated with bile leakage, one with lymphatic leakage and four with pleural effusion, and they were cured by non-surgical treatment. There were no patients with postoperative hemorrhage, incision infection or hepatic failure. No mortality was observed. The mean postoperative hospital stay was(19±14)days. Conclusions Radiofrequency energy was applied along the margins of the tumor to create zones of necrosis before resection with a scalpel, offering hepatobiliary surgeons an additional method for performing liver resections with minimal blood loss, low morbidity and mortality rates. As for malignant tumors, minor or major liver resection assisted by Habib 4X is safe, and it can reduce the chance of positive incisal margin.

Objective To observe the effects ofXiangsha Liujunzi Decoction (XSLJZ) on levels and mRNA of IL-6, IL-10 and protein expression of HSP70 of gastric mucosa in chronic atrophic gastritis (CAG) rats with spleen-stomach deficiency; To discuss its mechanism.Methods Rats were divided into 2 groups through random number table: normal group and model group. The model of CAG rat with spleen-stomach deficiency type was induced by synthetic methods. After successful modeling, rats were divided into model group, positive control group, XSLJZ high-, medium-, low-dose group. Rats in normal and model group received distilled water 10 mL/(kg?d) for gavage; XSLJZ high-, medium-, low-dose group received XSLJZ 24, 12, 6 g/(kg?d), respectively; positive control group received mycin 0.30 g/(kg?d) for gavage for 120 consecutive days. Generally living conditions, levels and mRNA of IL-6, IL-10 and protein expression of HSP70 in gastric mucosa tissue were detected by protein immunoblotting.Results Compared with the normal group, generally living conditions of rats in the model group were poor; mRNA and the content of IL-6 increased significantly, and mRNA and the content of IL-10 decreased significantly (P<0.05,P<0.01); mRNA and protein expression of HSP70 in gastric tissues was much lower than that of normal group (P<0.05,P<0.01). Compared with model group, generally living conditions of rats in the XSLJZ high-dose groups were improved significantly; mRNA and the content of IL-6 decreased significantly, and mRNA and the content of IL-10 increased significantly (P<0.05); XSLJZ high-, medium-dose groups mRNA and protein expression of HSP70 in gastric tissues increased significantly(P<0.05).Conclusion XSLJZ has protective effects on gastric mucosa of CAG rats with spleen-stomach deficiency.

"Symptom set" was changed into "combined set of syndrome factors" by exploring the mapping relationship be-tween symptom and syndrome factors,among which the ones meeting the definition of pathogenesis were chosen to further form the"pathogenesis set" under the ISOR combination principle,so as to build a new syndrome factor system of"symptom collection-syndrome factor collection-pathogenesis collection",which is not only beneficial to perfect the syndrome factor sys-tem,but also a new thought for applying mathematics and computer science to Chinese medicinal research.

The DNA coding for the fusion protein of thromobopoietin mimetic peptide (TMP) and human IgG1 Fc fragment was amplified from recombinant plasmid pET28a/TMPFc, inserted into pPICZalphaA and transformed into Pichia pastoris using electroporation. The recombinants of correct phenotype were identified after screening on MDH and MMH culture medium. The fusion gene was verified with PCR and western blot. MTT method was used to test the activity of TMPFc in promoting the growth of Ba/ F3-mpl cell. The TMPFc with a 64 000 molecular weight was a secretary protein in the system and its expression amounted to 65% of the total protein in the medium supernatant. The TMPFc showed a promotive effect on the growth of Ba/F3-mpl in vitro. A significant portion of the secretary protein existed as dimer, which provided material for studying the dimer in future.
Amino Acid Sequence ; Blotting, Western ; Humans ; Immunoglobulin Fc Fragments ; genetics ; Immunoglobulin G ; genetics ; Molecular Sequence Data ; Pichia ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; biosynthesis ; Thrombopoietin ; genetics

OBJECTIVETo investigate the effect of hepatitis C virus (HCV) strain JFH1 on expression of the human gene, growth arrest and DNA damage-inducible gene 45 alpha (GADD45a), in infected hepatoma cells.

METHODSHCV JFH1 RNA-containing supernatants were used to infect the human hepatoma cell line, Huh7.5.1; infection was confirmed by Western blot detection of the HCV-encoded non-structural 5A (NS5A) protein and core protein. Infection-induced changes in GADD45a mRNA and protein expressions were measured by real time PCR using SYBR Green and Western blotting, respectively. Significance of differences between the levels detected in JFH1-infected or uninfected Huh7.5.1 cells was analyzed by single factor analysis of variance testing.

RESULTSThe HCV infection system was successfully established, as evidenced by expression of NS5A protein and core protein. The GADD45a mRNA and protein levels were significantly down-regulated in JFH1-infected Huh7.5.1 cells, by 0.57+/-0.09 and 0.28+/-0.03, respectively, as compared to levels in uninfected Huh7.5.1 cells (F values were 75.407 and 560.04, respectively; P less than 0.01).

CONCLUSIONHCV inhibits the mRNA transcription and protein expression of host GADD45a, which may contribute to the pathogenesis of hepatocellular carcinoma caused by HCV infection.

Cell Line, Tumor ; DNA Damage ; Hepacivirus ; classification ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Transcription, Genetic

According to previous studies of SARS-CoV (Severe acute respiratory syndrome coronavirus), a variety of novel accessory genes have been identified in SARS-CoV genome, which were interspersed the structural genes of SARS-CoV and considered to be unique to the SARS-CoV genome. The predicted unknown proteins (PUPs) encoded by the accessory genes might play important roles in the SARS-CoV infection. Three of those genes, called X4, X5 and ORF10, were synthesized and introduced into E. coli to induce expression. SDS-PAGE and Western blotting revealed that the three genes have been expressed in E. coli. The induction of SARS PUPs genes expression in different temperatures, induction times, IPTG concentrations and A values of E. coli cells were performed. The optimal induction condition of SARS-CoV PUPs genes was characterized according to the orthorgonal analysis. The ratio of recombinant proteins of PUPs to total proteins is as follows: X4, 20%; X5, 27.8%; ORF10, 68.5% under the optimum conditions.
Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Viral ; Genes, Viral ; Genetic Vectors ; Genome, Viral ; Open Reading Frames ; Recombinant Proteins ; genetics ; metabolism ; SARS Virus ; genetics ; Temperature ; Time ; Viral Matrix Proteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism

OBJECTIVETo observe the dynamic time-phase expressions of key genes of brain-gut CaM signal pathway of spleen Qi deficiency rats and the intervention effect of Sijunzi decoction.

METHODMale Wistar rats were randomly divided into the normal control group, model 14 d, 21 d, 28 d groups, and Sijunzi decoction 14 d, 21 d, 28 d groups. Except for the normal control group, the remaining groups were included into the spleen Qi deficiency model with the bitter cold breaking Qi method (ig 7.5 g · kg⁻¹ · d⁻¹ of Rheum officinale, Fructus aurantii immaturus, Magnolia officinalis preparation) and the exhaustive swimming method. On the 7th day after the modeling, the Sijunzi decoction groups were orally administered with Sijunzi decoction 20 g · kg⁻¹ · d⁻¹. The expressions of key genes CaM/CaMK II of CaM signaling pathway in hippocampus and intestine at different time points by immunohistochemical method and Western blot. At the same time, the intervention effect of Sijunzi decoction on spleen Qi deficiency rats and its mechanism were analyzed.

RESULTSpleen Qi deficiency rats showed higher intestinal CaM/CaMK II expression and lower hippocampus CaM/CaMK II expression than normal rats (P < 0.05, P < 0.01). After the treatment of Sijunzi decoction, spleen Qi deficiency rats showed reduction in intestinal CaM/CaMK II expression and increase in hippocampus CaM/CaMK II expression (P < 0.05, P < 0.01).

CONCLUSIONThe formation of spleen Qi deficiency syndrome may be related to the high expression of CaM/CaMK II in small intestine tissues and its low expression in hippocampus tissues. Sijunzi decoction may achieve the therapeutic effect in spleen Qi deficiency syndrome by reducing the CaM/CaMK II expression in intestinal tissues and increasing it in hippocampus tissues.

Animals ; Brain ; drug effects ; enzymology ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; genetics ; metabolism ; Calmodulin ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Intestines ; drug effects ; enzymology ; metabolism ; Male ; Qi ; Rats ; Rats, Wistar ; Spleen ; drug effects ; Splenic Diseases ; drug therapy ; enzymology ; genetics ; metabolism