This study was aimed to establish a HPLC method for the content determination of liposoluble components, such as dihydrotanshione I, croptotanshinone, tanshinone I and tanshinone IIA, as well as the content determination of water-soluble components, such as danshensu, protocatechuic aldehyde, rosmarinc acid, salvianolic acid B, and salvianolic acid A in Danshen capsule simultaneously. For liposoluble components, the determination was performed on a Welch ultimate XB-C18 column (250 mm × 4.6 mm, 5 μm) by gradient elution using acetonitrile-water as the mobile phase. The flow rate was 1 mL·min-1. And the detection wavelength was set at 270 nm. For water-soluble components, the determination was performed on a Thermo Syncronis C18 column (250 mm × 4.6 mm, 5μm) by gradient elution using methanol-acetonitrile-0.02%phosphoric acid as the mobile phase. The flow rate was 1 mL·min-1. And the detection wavelength was set at 286 nm. The results showed that there were good linear relationships between components of peak areas and the ranges were 0.472-9.44 μg (r = 0.999 8) for danshensu, 0.352-7.04 μg (r = 0.999 9) for protocatechuic aldehyde, 0.244-4.88 μg (r = 1.000 0) for rosmarinc acid, 2.268-45.36 μg (r = 0.999 9) for salvianolic acid B, 0.168-3.36 μg (r = 0.999 9) for salvianolic acid A, 0.088-1.76 g(r=0.999 9) for dihydrotanshione I, 0.18-3.6μg (r=0.999 9) for croptotanshinone, 0.208-4.16μg (r=0.999 9) for tanshinone I, and 0.17-3.4μg (r=0.999 9) for tanshinone IIA. Average recoveries of the method were between 97.48%and 98.59%. It was concluded that the analysis was stable and reproducible, which can be used as a method for the analysis of Danshen capsule.

Protein-protein binding is the basis of virus and host cell interactions. With the application of technology of studying protein interactions, more knowledge of replication and pathogenesis of hepatitis C virus (HCV) was acquired. Non-structure protein 5B(NS5B) of HCV is a kind of viral protein, which plays an important role in replication of HCV. However, the effect of NS5B is not clear. To investigate the biological function of NS5B, we performed yeast two hybrid to look for proteins in hepatocytes interacting with NS5B. We constructed NS5B bait plasmid by cloning the gene of NS5B into pGBKT7, then transformed it into yeast AH109(a type). The transformed yeast was mated with yeast Y187(? type)containing liver cDNA library plasmid in 2?YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-?-gal for screening. Thirty-three colonies were selected and sequenced. Among them, two colonies were new genes with unknown function. The preliminary successful cloning of gene of protein interacting with NS5B paved the way for the study of the physiological function of NS5B and its associated protein.

Objective To screen the HCV NS4A binding protein. Methods By using HCV NS4A as a solidified selective molecule, the T7 select human liver cDNA library was biopanned and the positive clones were selected. After screening, the positive plaques was amplified and then cloned into the pGEM-Teasy vector. Two positive plaques were chosen for DNA sequencing. Results The binding protein of HCV NS4A was identified as mitogen-activated protein kinase (MAPK)-activated protein kinase 5 (MAPKAPK5) by BLAST. Conclusion This approach provides a new way for the study of the pathogenic mechanism of HCV infection.

Objective To screen the HBV core promoter binding protein, and to investigate their potential role in the replication of HBV DNA. Methods By using HBV core promoter being used as a selective molecule, the T7select human liver cDNA library was biopanned and the positive clones were selected. Results After phage display screening, the positive plaques was amplified and then cloned into the pGEM-Teasy vector. Six positive plaques were chosen for DNA sequencing. The binding protein of HBV core promoter was identified as caboxypeptidase N(CPN) by BLAST. Conclusion The results suggest that phage display screening of binding protein of HBV core protein provides a new approach to study the replication mechanism of HBV DNA.

Objective To express soluble human anti-idiotypic single chain Fv to hepatitis C core protein in E.coli. Methods Using phage display technique, the semisynthetic phage library was panned by HCV core monoclonal antibody which was coated in a microtiter plate. After three rounds of biopanning, 53 clones were identified specific to HCV core antibody. The specificity of anti-idiotypic scFv was determined by ELISA. After digested with Sfi/Not, the selected HCV core anti-idiotypic scFv positive clone was subcloned into the vector pCANTAB5E for the expression of E-tagged soluble anti-idiotypic scFv. The E.coli XL1-Blue was transformed and induced by IPTG. The specificity of anti-Id scFv was evaluated with ELISA. Results HCV core anti-Id scFv DNA digestion and sequence data showed that the scFv gene was composed of 774bp. ELISA results demonstrated that the soluble human HCV core anti-idiotypic scFv to HCV core monoclonal antibody had a specific combination character. The molecular weight of expressed HCV core anti-idiotypic scFv was 28kD as shown by SDS-PAGE. Conclusion HCV core anti-Id scFv has been successfully expressed in E.coli.

Objective To clone the human target gene HBVDNAPTP1 transactivated by hepatitis B virus DNA polymerase obtained by screening with suppression subtractive hybridization (SSH) and bioinformatics techniques. To construct prokaryotic expressive vector of HBVDNAPTP1 gene, induce the expression of recombinant protein in E. coli, and analyze the expression level of HBVDNAPTP1 gene in human tissues. Methods The DNA fragment of HBVDNAPTP1 was amplified by reverse transcription polymerase chain reaction (RT-PCR) taking mRNA from HepG2 cells as the template, and the correct DNA fragment was then inserted into inducible prokaryotic expressive vector pET-32a (+). The competent BL21 (DE3) E. coli was transformed, and then cultured and induced with IPTG. The expressed HBVDNAPTP1 was confirmed with Western blot. UniGene database was used to analyze the chromosome mapping and tissue expression profile of HBVDNAPTP1 gene. Results The DNA fragment of HBVDNAPTP1 was amplified by RT-PCR. HBVDNAPTP1 expressive vector was constructed. After transformation with pET-32a(+)-HBVDNAPTP1 and induction with IPTG, recombinant HBVDNAPTP1 was expressed and confirmed by Western blot. The expression of genomic location of HBVDNAPTP1 gene was low in multiple-tissues with the exception of pituitary gland, tonsil, tongue, thymus, trachea and umbilical cord. Conclusion The recombinant HBVDNAPTP1 gene could be expressed in prokaryotic expression system of E. coli. The chromosome mapping and tissue expression level of HBVDNAPTP1 gene is tentatively conceived.

Objective To study the exact function of HBeBP4A so as to investigate the gene expression of HBeBP4A in yeast cell.Methods Reverse transcription polymerase chain reaction(RT-PCR)was employed to amplify the gene of HBeBP4A from recombinant plasmids pcDNA 3.1/myc-HisA-HBeBP4A,and the gene was cloned into pGEM-T vector.The gene of HBeBP4A was cut from pGEM-T-HBeBP4A vector and cloned into yeast expressive plasmid pGBKT7,and pGBKT7-HBeBP4A was then transformed into yeast AH109.The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting hybridization.Results HBeBP4A gene was successfully amplified and identified by DNA sequencing.The digested fragment was cloned into pGBKT7 vector and transformed into yeast cell AH109.The SDS-PAGE and Western blotting assay showed that the relative molecular weight of the expressed product was about 61.37kD,and HBeBP4A protein existed in yeast cells.Conclusion The findings suggested that HBeBP4A was successfully expressed into yeast system.

Objective To study the interaction between HCV core protein and HCBP6 in mammlian cells using CheckMateTM Mammalian Two-Hybrid System.Methods cDNA fragments encoding HCV core protein and HCBP6 were amplified by PCR and subsequently cloned into pGEM-T vector.After verified by sequencing,the target fragments were subcloned into mammalian two-hybrid plasmids,pBIND and PACT,respectively.The recombinant plasmids,pBIND-core and pACT-HCBP6 were co-transfected into HepG2 cells with reporter plasmid pG5luc.pBIND+pACT were induced as background controls,pBIND-Myod+pACT-Id as positive controls,and pBIND-core+pACT,pBIND+pACT-HCBP6 as blank controls.The expression of G5luc,which indicated the interaction between HCV core protein and HCBP6 in mammlian HepG2 cells,was assayed through Dual-Luciferase Report Assay System and Turner Biosystems Veritas Microplate Lunimometer.Results The recombinant vectors pBIND-core and pACT-HCBP6 were successfully constructed.When co-transfected into HepG2 cells with reporter plasmid pG5luc,there were significant differences in the luciferase activity in the pBIND-core and pACT-HCBP6 groups compared with every control group.Conclusions HCBP6 can interact with HCV core protein in HepG2 cells,which provides clues for further study on the function of HCBP6 and core proteins,and on the mechanism of HCV core in cell apoptosis and cancer transformation.

Objective To explore the feasibility and reliability to build a sinoatrial node damage model in canine induced by formaldehyde wet dressing.Methods Twenty dogs were randomly divided into 4 groups(5 in each group) by means of random number table:2-hour,24-hour,1-week and 4-week groups after wet dressing.The sinoatrial node area of canine was damaged by wet dressing with 20% formaldehyde.Sinus node function was measured before,2 h after wet dressing and corresponding times of each group.Electrocardiogram(ECG) of body surface were recorded synchronically.Results Average wet dressing time was 6.2?2.6(3 to 12)min.Five dogs showed significantly decrease of heart rate(HR)(140?11 vs 89?6 beat/min,P

Objective To evaluate the importance of clinical screening and follow up by direct colonoscopy for colorectal cancer at an early and curable stage. Methods There were 2 196 elderly people aged between 60 to 89 years. The clinical screening by direct colonoscopy was performed according to the protocol. 1 740 of 2 196(79.2%) patients were followed up every year. Results Fifty two elderly persons were found to be colorectal cancer patients by colonoscopy, with the detectable rate being 2.4%. Nineteen were diagnosed early stage colorectal cancer, accounting for 36.5% of the detected colorectal cancer. Nine among the followed up cases were detected early colorectal cancer, accounting for 45 0% of the detected colorectal cancer. The resectable rate and the 5 year survival rate was 97 7% and 80 9% for colorectal cancer, respectively. 98 9% of the cecum intubation cases was successful. The incidence of complication for colonoscopy was 0 05%. Conclusions By clincal colonscopy screening and follow up study for colorectal cancer and precancerous changes in the elderly, the patients with adenomatoid polyps were early diagnosed and treated, so it raised the detectable rate of early colorectal cancer and the level of grade prevention of colorectal cancer.