In order to find out micro-spore extracting method suitable for transcriptome sequencing,this study took Chinese cabbage‘Jihong 82’as experimental material and micro-spore induced with heat shock as research object.This study compared and analyzed the RNA extraction effects by 3 different methods:TRIzol method,improved CTAB method and ultrapure kit method.The results showed that RNA concentrations extracted by TRIzol method and improved CTAB method were higher,but the RNA integrity was lower.Quality of RNA extracted by ultrapure kit method was the highest,and the RNA inegrity was also good.Therefore,RNA obtained by this method was suitable for satisfying the requirement of transcriptome sequencing.

Objective:To prepare monoclonal antibodies(McAbs) against NS1 protein of West Nile virus(WNV-NS1).Methods:BALB/c mice were immunized intraperitoneally with the purified NS1 protein expressed by prokaryotic expression system.Then the spleen cells of immunized mouse were fused with SP2/0 myeloma cells by the PEG regent.Meanwhile,an indirect ELISA using the purified NS1 protein derived from eukaryotic expression system as antigen was developed to screen antibody-producing hybridomas.Western blot and IFA were applied to characterize the McAbs.Results:Two hybridoma cell strains secreting McAbs against WNV-NS1 protein,designated as WN-1C10 and WN-3D10,were obtained.Conclusion:The results of Western blot and IFA indicate that these two McAbs are specific for WNV-NS1 protein,and don't cross-react with Japanese encephalitis virus(JEV),so they are helpful to develop differential diagnosis between WNV and JEV.

In this study,the M gene of Porcine reproductive and respiratory syndrome virus(PRRSV) CH-1a strain was amplified from the PRRSV genome by RT-PCR and cloned into the plasmid pSC11.The recombinant plasmid pSC11-PRRSV-M was transfected into TK-143 cells infected with vaccinia virus WR strain.Recombinants vaccinia virus of rWR-PRRSV-M was obtained by selecting blue plaques on the TK-143 cells overlaid with agar containing X-gal,and confirmed by PCR.The M gene expressed in TK-143 cells were identified by SDS-PAGE and confirmed by immunofluorescence assay(IFA).The rWR-PRRSV-M recombinant virus could induce specific humoral immune responses against PRRSV in the immunized mice.

The NS1 protein of West Nile virus NY99 strain was amplified by PCR with specific primers and cloned into pMAL-c2X.The recombinant vector was transformed into E.coli TB1 competent cells and a fusion protein MBP-NS1 was expressed after induction with IPTG.The MBP-NS1 protein was purified and analysed by western blot and indirect enzyme-linked immunosorbent assay.The results showed that the purified fusion protein retained good antigenicity and specificity.

The GP5 gene of porcine reproductive and respiratory syndrome virus(PRRSV) CH-1a strain was amplified from the PRRSV genome by RT-PCR,and cloned into the transfer plasmid pSC11 to produce pSC11-GP5.Recombinant vaccinia virus containing the GP5 gene(rWR-PRRSV-GP5) was generated through homologous recombination between pSC11-GP5 and vaccinia virus WR strain in TK-143 cells,purified by plaque assay and identified by PCR.Expression of the GP5 protein was detected by SDS-PAGE.Antigenicity of the recombinant GP5 protein was confirmed by indirect immunofluorescence assay(IFA).Inoculation to mice with rWR-PRRSV-GP5 induced specific humoral immune responses against PRRSV.

OBJECTIVE:To express the fusion protein of glutathione S-transferase (GST) and human Id-2 in E. coli and prepare the polyclonal antibodies against Id-2.METHODS:The coding sequence of Id-2 gene was amplified by RT-PCR from the total RNA of breast cancer tissue. The recombinant plasmid was identified by PCR, restriction endonuclease digestion analysis and sequencing. The fusion protein GST-Id-2 expressed in E. coli following IPTG induction was purified by glutathione-agarose affinity chromatography and used to immunize rabbits to prepare the polyclonal antibodies against GST-Id-2.RESULTS:PCR, restriction endonuclease digestion and sequence analyses showed that the Id-2 gene had been correctly inserted into pGEX-6P-1 vector, and the GST-Id-2 fusion protein expressed had a relative molecular mass of approximately 40,000 as shown by SDS-PAGE. The polyclonal antibodies obtained from the rabbit sera were found to specifically react with purified Id-2 by Western blotting, ELISA and agar gel immunodiffusion (AGP).CONCLUSION:The prepared polyclonal antibodies against Id-2 allow effective Id-2 detection and facilitate further investigation of the structure and antigen epitope of Id-2.

To examine the bioactivity of recombinant equine interleukin-18 (rEIL-18) obtained from Escherichia coli, rEIL-18 was evaluated to see if it showed a synergistic effect with recombinant human interleukin-12 (rhIL-12) for the induction of equine interfer-on-γ (EIFN-γ ) gene expression from equine peripheral blood mononuclear cells (PBMCs) by Real-time RT-PCR. REIL-18 could in- duce EIFN-γ mRNA expression from equine PBMCs about 20-fold higher than that of negative control in the presence of rhIL-12. And the effect to induce EIFN-γ gene expression was dose-dependent with rEIL-18 in a fixed concentration of rhIL-12. Nine an-ti-rEIL-18 monoclonal antibodies (mAbs) were also evaluated if they could neutralize the bioactivity of rEIL-18. And one of the 9 mAbs could neutralize rEIL-18 bioactivity and its effect was dose-dependent.

With the development of biomedicine,there are an increasing number of ethical conflicts arising from animal experiments.Laboratory animal science,which uses experiment animals and animal experiments as its research objects,may relieve the conflict.We adopted various teaching patterns and introduced the internationally accepted 3R principle(refinement,reduction and replacement) into the teaching of Laboratory Animal Science course,which has obtained satisfactory results.