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To prepare monoclonal antibody (mAb) against CP4-EPSPS, which could be applied to the development of gold colloidal rapid diagnostic kit for the specific detection of GMO.BALB/c mice were immunized with CP4-EPSPS. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells. The hybridoma cells that secreted CP4-EPSPS mAb antibodies were cloned with limited dilution method. The immunoglobulin (Ig) subtype, the ascites titers, the binding site, and the affinity of the obtained mAbs were determined by indirect ELISA. The specificity of mAbs was tested by ELISA and Western blot analysis.From over 80 positive hybridomas which secreted anti-cp4-EPSPS mAbs, a pair of hybridomas were screened out, designated III5A3 and III13A2. Chromosome analysis revealed that the obtained hybridomas were with the universal characteristics of the monoclonal hybridoma cells which secreted mAb, and the Ig subtype of III5A3 and III13A2 mAb was both IgG1. The ascites titers of III5A3 and III13A2 mAb were 1:10(6) and 1:10(8), respectively. Our study also demonstrated that these two mAbs, which recognized the same epitope, could both specifically bind to the CP4-EPSPS protein. The relative affinity constant of III5A3 and III13A2 mAb was determined as 10(5) and 10(6) respectively.A pair of high titer, specific mAbs against CP4-EPSPS have been successfully prepared and primarily identified, which may be useful in the development of a rapid and convenient diagnostic kit for detection of GMO.

To prepare monoclonal antibody (mAb) against Vibrio cholerae O139, which would be used as the gold colloidal reagent strip for rapid detection of O139, and to determine its biological characterization.BALB/c mice were immunized by inactivated Vibrio cholerae O139. Anti-O139 mAbs were prepared by using hybridoma technique. The specificity of mAb was determined by indirect ELISA and Western blot. The indirect ELISA was used to identify Ig subgroup, detect its titer in ascites and relative affinity, and analyze antigen-binding epitope of mAbs.Two hybridoma cells (O4D7 and O4D10), secreting anti-O139 mAbs were obtained. The Ig subgroups of O4D7 and O4D10 were IgG2b and IgG3, respectively. The mAb's titer in ascites was 1:10(7). The relative affinity of mAb O4D7 was more than 10(5) and that of O4D10 was more than 10(4). The result of additive ELISA showed that two mAbs could recognize different antigen epitopes.The successful preparation of two anti-O139 mAbs provides a powerful tool for the development of a rapid method of detecting Vibrio cholerae O139 infection.

To construct and express fusion gene encoding Hyper-IL-6 in mammalian cells.Using geneSOEing method, human sIL-6R cDNA was fused with IL-6 cDNA by a linker rich in glycine through PCR technique and cloned into pIRES(2)-EGFP. The constructed plasmid was transfected into mammalian cells. The expression of fusion gene encoding Hyper-IL-6 was detected by GFP and RT-PCR and immunocytochemistry.The recombinant plasmid pIRES-HIL-6 was successfully constructed and expressed in mammalian cells.The success in construction and expression of human Hyper-IL-6 makes it possible to further study its biological functions.

To construct a recombinant vector containing fused gene of heat shock protein A(HspA) and outer membrane protein(OMP) with M(r) 18,000, from human Helicobacter pylori(Hp) and express the fusion protein in E.coli BL21.The gene encoding HspA was amplified from Hp chromosome by PCR. After digestion with kpn I and BamH I, the HspA gene was inserted into the prokaryotic expression vector pET32a(+). After recombinant vectors pET32a(+)/HspA and pET32a(+)/Omp (18) were digested with Hind III and BamH I, the pET32a(+)/HspA and 18,000 OMP gene segments were recovered through agarose electrophoresis, and connected by T4 ligase. The recombinant vector pET32a(+)/HspA-Omp(18) was transformed into E.coli BL21(DE3). The antigenicity of recombinant fusion protein was analysed by Western blot.Enzyme digestion analysis and sequencing showed that the fused gene had 891 base pairs. As compared with gene reported in GenBank, there were 1.15% and 1.26% differences in cloned nucleotide sequence and amino acid sequence respectively. SDS-PAGE analysis showed that recombinant vector could be expressed in E.coli BL21. The relative molecule mass (M(r)) of expressed fusion protein was 51x10(3). And soluble expression product accounted for 18.96% of total bacterial protein.After purification via Ni-NTA agarose resin, the purity of recombinant fusion protein was about 95%. The Western blot analysis showed that recombinant fusion protein could be recognized by anti-Hp positive serum and anti-18,000 OMP mAb, suggesting that this protein had good antigenicity.The fused gene of HspA and OMP is cloned and expressed successfully, which lays the foundation for development of protein and DNA vaccines and a diagnostic kit of Hp infection.