Objective To express the recombinant antigen coded by Pagumogonimus skrjabini adult cysteine protease gene fragment and to investigate the immunoreactivity of the fusion protein for further usage on serodiagnosis of the fluke disease. Methods The target gene fragment was amplified by PCR. After purification with gel purification recoverykit, the target gene fragment was ligated with PinPointTM Xa 1 T vector and transduced into E.coli JM109 strain. The expressed fusion protein sample was prepared with alkaline lysis solution, and then analyzed by SDS PAGE. The expression level was determined by Coomassie blue staining and the streptavidin alkaline phosphatase staining. The immunoreactivity of fusion protein was examined by Western blotting. Results A total of 8 positive clones were harvested, and only one had the proper orientation verified by sequencing. The recombinant antigen was obtained after being induced with IPTG (Isopropltio ? D galactoside), and a positive band of 32?10 3 was found by streptavidin alkaline phosphatase staining. In the same position, the fusion protein was also detected by Western blotting. Conclusion The expression vector of adult cysteine protease gene PinPointTM Xa 1 T vector was successfully constructed. The recombinant antigen obtained after being induced with IPTG possesses good immunoreactivity.

Objective To investigate the chemical constituents of Lappula echinata and determine the antibacterial activity.Methods A new quinolone alkaloid was isolated from the BuOH extract of L.echinata by silica gel column chromatography and gel column chromatography.Its structure was identified by 1H-NMR, 13C-NMR, 2D-NMR, HR-MS, UV, and IR spectral data analysis.Its antibacterial activity was determined by KB method.Results A new quinoloe alkaloid named 8-methoxy-4-quinolone-2-caboxylic acid was isolated from L.echinata and was found to have antibacterial activity on Pseudomonas pyocyanea ATCC 27853, EPEC O111, pneumobacillus and Staphylococcus epidermidis.Conclusion This is a new compound with antibacterial activity.

Objective To study the diterpenoids possessed immunosuppressive activity from Tripterygium hypoglaucum. Methods The chemical constituents were isolated from the CHCl3 extracts of T. hypoglaucum by repeated column chromatography, including silica gel, Sephadex LH-20, and preparative HPLC. Their structures were elucidated on the basis of spectroscopic studies. The immunosuppressive activity of these compounds was tested using the lymphocyte transformation test. Results Compounds Ⅰ-Ⅵ were identified as triptobenzene H (Ⅰ), triptoquinone A (Ⅱ), triptoquinone B (Ⅲ), triptoquinone H (Ⅳ), triptonediol (Ⅴ), triptonoterpene (Ⅵ). Conclusion Compound Ⅰ-Ⅲ are isolated from this medicinal plant for the first time and all the compounds show the significant immunosuppressive activity.

Objective To study the anti-cancer constituents from Tripterygium wilfordii. Methods Chemical constituents were isolated by repeated column chromatography (silica gel, Toyopearl HW-40 C and preparative HPLC), their structures were elucidated on the basis of spectroscopic methods, and the anti-cancer activity was screened by MTT method. Results Five diterpenes, 3-epi-triptobenzene B (Ⅰ), 3?, 14-dihydroxy-abieta-8, 11, 13-triene (triptobenzene B,Ⅱ), wilforol E (Ⅲ), triptohairic acid (Ⅳ), and 11-hydroxy-14-methoxy-18(4→3)-abeo-abietan-3, 8, 11, 13-tetraen-18-oic-acid (hypoglic acid, Ⅴ) were isolated from T. wilfordii. Conclusion Compound Ⅰ is a new compound named as triptobenzene L, compound Ⅳ is isolated for the first time. The compounds Ⅰ-Ⅴ show the positive anti-cancer effects on HeLa and L929 cell lines.

Objective To construct eukaryotic expression plasmid of human B7-H1 extracellular region-hIgG1Fc-pCI-neo eukaryotic expression vector,and express the functional fusion protein in mammalian CHO cell.Methods Full-length human B7-H1 encoding sequence was amplified from human activated T cell cDNA library by PCR,fused with hIgG1Fc,then transformed into pCI-neo expression vector and verified by sequencing.The validated recombinant was transfected into mammalian CHO cell by lipofectamine reagent.The supernatant of the cultured cell was collected and analyzed by the sandwhich ELISA to detect if there was the fusion protein,and the fusion protein was purified by HiTrap recombination protein Protein A affinity chromatography.The concentrated supernatant or purified fusion protein were used for Western blotting after SDS-PAGE to identify the molecular weight and immune activity of the fusion protein of B7-H1.Results The extracellular region of hB7-H1 about 727 bp was cloned from human T cell cDNA library and was inserted with hIgG1Fc into the eukaryotic expression vector pCI-neo.After the transfection of recombinant into mammalian CHO cell by lipofectamine reagent,the expression of B7-H1 fusion protein was detected in the cultured CHO cell supernatant by the sandwhich ELISA.The immune activity of the fusion protein was verified by Western blotting,and its molecular weight was about 51.76?10~(3),very close to the expected value.Conclusion The hB7-H1-Fc chimeric molecule was successfully constructed and the expression of its functional fusion protein in mammalian CHO cells lays a foundation for further research on the role of B7-H1 in immune tolerance,autoimmune diseases.

Objective To establish a calculation software to determine paternity index(PI) in parentage testing and likelihood ratio (LR) in individual recognition.Methods Based on relevant industry standards and literature, using Visual Basic 6.0 to write the program.Results We successfully developed the calculation software for paternity index (PI) in parentage testing and likelihood ratio (LR) in individual recognition.Conclusion The calculation software can help staff to improve the calculation efifciency, and serve the forensic evidence.

Objective To develop an HPLC method for determination of triptoquinone H in Tripterygium wilfordii and its Tablet preparations. Methods An external method with Agilent Zorbax SC-C_(8)(250 mm?4.6 mm, 5 ?m) column as fixed phase and methanol-water (75∶25) as mobile phase was adopted. The detective wavelength was 258 nm and the flow rate was 1.0 mL/min. Results The linearity range of triptoquinone was 6.28 — 100.5 ?g/mL (r=0.999 7). The average recovery of T. wilfordii was 96.94%, RSD was 1.57% (n=9) and the average recovery of T. hypoglaucum Tablet was 100.02%, RSD was 1.74% (n=9). Conclusion The method is accurate and sensitive. It is adoptable for quantity analysis of triptoquinone H in T. wilfordii and its Tablet.

Objective To determine the content of tripterine in Tripterygium wilfordii and its tablets by HPLC.Methods An external standard method by HPLC with Zorbax C_(18)column as fixed phase and methanol-1% HAc(87∶13) as mobile phase was adopted.The detection wavelength was 425 nm and the flow rate was 1.0 mL/min.Results The linear range for tripterine was 40.96～204.8 ?g/mL(r=(0.999 6).) The average recovery of Chinese medicinal materials was 98.37% and RSD was 1.01%(n=9);the average recovery of preparation sample was 98.59% and RSD was 1.18%(n=9).Conclusion The method is simple and accurate,which can be adoptable for quantitative analysis of tripterine in the plants of Tripterygium L.