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Brucella cell surface protein (BCSP31) is potentially useful for diagnosing brucellosis. We aimed to establish a monoclonal antibody (MAb) against Brucella melitensis BCSP31 and to investigate its distribution in diagnosis. Soluble recombinant BCSP31 was successfully expressed and purified. Two MAbs (1F1 and 1E5) against B. melitensis BCSP31, effective in detecting both recombinant and cellular proteins, were obtained and characterized. The MAbs did not react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus aeruginosus, but strongly reacted with BCSP31 and B. melitensis by ELISA and Western blot analysis. We also tested different Brucella species and brucellosis using the prepared anti-BCSP31 MAbs. BCSP31 and anti-BCSP31 MAbs may play important roles in future research in diagnosing brucellosis.

To express G2 fragment of M segment and 0.7 kb fragment of S segment and several CTL epitopes of S segment in adenovirus expression system and investigate the immunological properties of hantaan virus chimeric gene.The recombinant adenovirus was constructed and the recombinant adenovirus was obtained after transfecting HEK293 cells. The titer of it was determined and the expressed product was detected by IFA and ELISA. Further, BALB/c mice were vaccinated by the recombinant adenovirus and the immune response was tested by ELISA, microcell-culture neutralizing experiment, T lymphocyte proliferation test (MTT assay) and cell-mediated cytotoxicity assay.The recombinant adenovirus AG2S0.7CTL1, AG2S0.7CTL2 were constructed successfully and the titer of it was about 10¹⁰-10¹¹ pfu/mL. The expressed protein could be recognized by the hantaan virus NP-specific mAb and glycoprotein G2-specific mAb. The recombinant adenovirus containing CTL epitopes could elicit effectively the cellular immune response aimed to the NP and GP of hantaan virus in BALB/c mice.The recombinant adenovirus containing CTL epitopes could induce the higher cellular immune response than the group that not containing CTL epitopes.

To construct a adenovirus vector containing the 0.7 kb fragment of S gene of Hantavirus, CAG promoter, and WPRE (mRNA-stabilizing post-transcriptional regulatory element from the woodchuck hepatitis virus).The fragments of CAG and WPRE were synthesized according to GenBank, and inserted into the plasmid pShuttle-S0.7 to create a transfer vector pShuttle-S0.7-CAG-WPRE. The S0.7-CAG-WPRE fragment was then cloned into Adeno-X; Viral DNA by PI-Sce I and I-Ceu I digestion. The recombinant adenovirus DNA was linearized by Pac I, transfected into HEK 293 cells via Lipofectamine; 2000, and the titer of the recombinant adenovirus was determined by Adeno-X; Rapid Titer Kit. The expressed product of S0.7-CAG-WPRE fragment was detected by immunofluorescence assay.The sequence of S0.7-CAG-WPRE fragment was confirmed by sequencing, and the recombinant adenovirus containing S0.7-CAG-WPRE was titered at 10(13); pfu/L. HEK293 cells transfected with recombinant adenoviruses were detected positive by immunofluorescence assay using a specific mAb 1A8 against Hantavirus nucleoprotein.The recombinant adenovirus containing the 0.7 kb fragment of S segment of Hantavirus, CAG promoter, WPRE was constructed.

To construct the transgenic Arabidopsis thaliana containing full-length gene of mouse/human chimeric antibody(3G1MH) against Hantaan virus.The recombinant plasmid 3G1MH-pCAMBIA2301 was transformed into Agrobacterium tumefaciens GV3101 by TSS freeze-thaw method, and then the recombinant was transferred into wild Arabidopsis thaliana by vacuum-transgenic method. The regenerated transgenic plants were selected with kanamycin, and confirmed by PCR and Northern blot.PCR result showed stable integration of the 3G1MH gene IN Arabidopsis thaliana genome in 7 stains of the transformed plants. Northern blot analyses confirmed the transcription of heavy and light chains in the transgenic plants.The successful establishment of 3G1MH transgenic Arabidopsis thaliana plants pave the way for further research on expressing therapeutic antibody in transgenic plants.

Survivin is a novel tumor-associated gene, its overexpression mostly associates with carcinogenesis and development. Nevertheless, the precise role of survivin in initiation and progression of gliomas is still not completely clear. We constructed here three short hairpin RNA (shRNA) targeting survivin plasmid vectors and introduced them into glioma U251 cells. The three shRNAs were efficiently and specifically able to knockdown the survivin expression in transiently transfected U251 cells. The stable transfectants expressing the shRNA having the strongest inhibitory effect against survivin exhibited decreased cell growth, increased spontaneous apoptosis, mitotic catastrophe and cell cycle arrest. Furthermore, in nude mice xenografts, the stable transfectants presented decreased de novo glioma formation and reduced development of angiogenesis. Results from this study indicate that survivin plays an important role in malignant proliferation, antiapoptosis and angiogenesis of gliomas, which may become an attractive target for gene therapy of gliomas, while RNA interference (RNAi) mediated by shRNA may become a new promising strategy for cancer gene therapy.

To investigate the effect of Hepatitis C virus (HCV) core protein on the expression of cyclooxygenase 2 (COX-2).The genes encoded HCV core protein were amplified from plasmid containing full length genome of HCV strain H77 using PCR, and were cloned into eukaryotic expression vector pcDNA3.1. The recombinant HCV-C/pcDNA3.1 was transiently co-transfected into HepG2 cells with luciferase reporter vector containing COX-2 promotor (COX2pro1.5 kb/luc). The luciferase activity and COX-2 protein expression were detected.The recombinants HCV-C/pcDNA3.1 have been constructed successfully. The luciferase activity of COX-2 promotor was activated by the expressed HCV core, and the increased protein expression of COX-2 in transfected HepG2 cells was detected by Western blot.HCV core protein can activate the COX-2 promotor and induce its expression, which provides a new experimental basis for further research on relationship between COX-2 and HCV pathogenesis.

To express hantaan virus(HTNV) envelope glycoprotein G(2) recombinant adenovirus(Adeno-G(2)) in vero E6 cells and explore its property of inducing immune response.Vero E6 cells were infected with the HTNV Adeno-G(2) (100 MOI). The expression of Adeno-G(2) in the infected Vero E6 cells was detected by IFA. BALB/c mice were immunized with HTNV Adeno-G(2), then the immune response to Adeno-G(2) was tested by ELISA, microcell-culture neutralizing experiment and lymphocyte proliferation test (MTT colorimetry).IFA detection showed the expression of Adeno-G(2) in the infected Vero E6 cells. The titer of specific antibody was 1:40; The low-titer neutralization antibody was also detected. But the lymphocyte proliferation reaction was not notable.The HTNV Adeno-G(2) can stimulate BALB/c mice to develop specific humoral immune response instead of specific cell-mediated immunity. This study provides the experimental basis for the development of gene engineering vaccine of HFRS.

To transiently express an intracellular single chain Fv of monoclonal antibody 1A8 against nucleocapsid protein of Hantavirus and characterize the immunological activities of the expressed products.COS-7 cells were transfected with mammalian expression vector 1A8-scFv-Ckappa/pCI-neo via lipofectin. The expressed product was identified by indirect immunofluorescence and immunoprecipitation.A diffuse pattern fluorescence was observed in less than 1% cytoplasm of transfected COS-7 cells. The binding of intracellular antibody fragments to NP antigen was confirmed by immunoprecipitation analysis.Transiently expressed single chain intrabodies can effectively target NP antigen in the cytoplasm. The present study may provide a new approach for treatment of Hantavirus.

To express the glycoprotein G2 and amino terminal of nucleoprotein (NP) of Hantaan virus in Bac-to-Bac baculovirus expression system in the form of fusion protein.The recombinant baculovirus expression vector pFBDHTa-G2S 0.7 was constructed. The chimeric gene was inserted into bacmid in E.coli DH10Bac with the help of Tn7 transposition system. Then the recombinant baculovirus was screened and the fusion protein was expressed in insect cells. The expression product was detected by ELISA, immunofluorescence assay and Western blot analysis.The recombinant baculovirus containing the chimeric gene G2S 0.7 had been constructed successfully and the fusion protein could be expressed in insect cells. The expressed protein could be recognized by the Hantaan virus NP-specific mAb and glycoprotein G2-specific mAb.The successful expression of fusion protein G2S 0.7 with biological activity in insect cells lays the foundation for further research on its immunological characteristic.