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Aim: To construct the vector stably expressing small hair RNA(shRNA) and investigate the effect of shRNA expressed with DNA vector on HBV replication and expression., Methods: The vector expressing shRNA designated pU6 was constructed by U6 promoter. The sequence of shRNA targeting on specific region of HBV genome against HBV was designed and synthesized, then cloned into pU6. The plasmid designated pU6B/HBVi was transfected into 2.2.15 cells using Lipofectamine 2000 reagent. The HBsAg and HBeAg in the supernatants of the transfected cells were measured by ELISA. The HBV copies in the transfected cells were measured by quantitative PCR., Results: Compared with controls, not only concentration of HBsAg and HBeAg but also HBV copies in transfected 2.2.15 cells decreased significantly by the plasmid expressing shRNA against HBV., Conclusion: HBV replication and expression in 2.2.15 cells are inhibited by stably expressed shRNA.

Aim: To construct the recombinant vector containing F1 gene caf1 of Y.pestis and express it in Saccharomyces cerevisiae., Methods: F1 gene caf1 of Y.pestis was inserted into pHSS6-mTn-3xHA/lacZ plasmid to construct recombinant vector pHSS6-mTn-3xHA/lacZ-caf1. The recombinant plasmid was linearized with Not I and then transformed into yeast cells by acetate lithium (LiAc) method. Positive recombinants were selected with uracil-lack medium. The expressed F1 antigen was identified by SDS-PAGE and Western blot., Results: SDS-PAGE and Western blot analysis showed that F1 antigen was expressed in Saccharomyces cerevisiae., Conclusion: The recombinant vector pHSS6-mTn-3xHA/lacZ-caf1 has been constructed and expressed successfully in Saccharomyces cerevisiae. These results lay the foundation for preparing gene vaccine of Y.pestis which could be taken via alimentary tract pathway.

Aim: To construct the phage antibody library for Fab fragment from a convalescent patient infected with SARS coronavirus., Methods: Total RNA was extracted from peripheral blood lymphocytes of a patient with SARS virus-specific IgG antibody and was transcribed reversely into cDNA. Using the cDNA as template, Fd and light chain of Fab genes were amplified by PCR with specific primer and were cloned into phagemid pComb3. Then the recombinant phagemid was electroporated into E.coli XL-1 Blue. The recombinant rate was detected by enzyme digestion analysis, and library repertoire was determined., Results: The antibody library of Fab fragment from a convalescent patient infected with SARS conronavirus was constructed. The recombinant rate of light chain and heavy chain of Fd gene were 91% and 75%, respectively. The library size was 7.23 x 10(7)., Conclusion: Phage antibody library for human Fab fragment has been constructed successfully, which lays the foundation for further study.

Aim: To express the recombinant angiogenin protein in mammalian cells., Methods: Human ANG full-length cDNA was obtained by chemical synthesis. The target gene ANG was inserted into eukaryotic expression vector pEGFP-C2. The recombinant plasmid was transfected into the human umbilical vein endothelial cells (HUVECs) via Lipofectin transfection. The expression of ANG gene in transfected HUVECs was detected by fluorescence microscopy and immunohistochemical staining., Results: DNA sequencing showed the sequence of the synthetic ANG was correct and amino acid sequence of ANG was right. The green fluorescence could be seen in transfected HUVECs under fluorescence microscope. Immunohistochemical staining detection showed that ANG expressed in transfected cells., Conclusion: Human ANG full-length cDNA has been obtained. The ANG protein was expressed in mammalian cells successfully.

Aim: To explore the effect of calcium ionophore (CI) on dendritic cells (DC) derived from peripheral blood monocytes., Methods: Peripheral blood monocytes from healthy donors were treated with 100 microg/L rhGM-CSF, 100 microg/L rhGM-CSF plus (10 microg/L) CI, and 100 microg/L rhGM-CSF plus (100 microg/L) respectively. After cultivation of 40 hours, cellular morphology was observed under light microscope and electron microscope. Surface markers on treated PBMCs were analyzed by flow cytometry. MTT colorimetry was used to detect proliferation of autologous T cells., Results: Peripheral blood monocytes treated with 100 microg/L rhGM-CSF plus 100 microg/L CI for 40 hours showed typical morphology of DCs. Simultaneously there was a decrease in CD14 expression and increase in HLA-DR, CD40, CD83 and CD86 expressions on these cells. In addition, PBMCs treated with 100 microg/L rhGM-CSF pass 100 microg/L CI for 40 hrs. Could evidently stimulate proliferation of autologous T cells., Conclusion: CI can markedly enhance transformation of peripheral blood monocytes induced by GM-CSF into DCs.