ABSTRACT
We transformed the fip-fve gene into Pichia pastoris GS115 for inducible and constitutive expression to obtain feasible bioactvie recombinant Fip-fve. The fip-fve gene was cloned from Flammulina velutipes fruting body by PCR and ligated to pPIC9 to construct inducible expression vector pPIC9-FIP-fve, and promotor pgap was used to replace the paox1 to construct constitutive expression vector pPIC9-PGAP-FIP-fve. These two vectors were used to transform P. pastoris by PEG method. The fip-fve was expressed after histamine-absence screening and yeast colony PCR. The inducible expression level reached 158.2 mg/L at the fourth day and the constitutive expression level was 46.3 mg/L and 29.5 mg/L using glucose and glycerol, respectively. The SDS-PAGE and Western blotting both proved the correctness of rFip-fve, and the hemagglutination test indicats the rFip-fve's bioactivity.
Subject(s)
Electrophoresis, Polyacrylamide Gel , Flammulina , Chemistry , Fungal Proteins , Genetic Vectors , Pichia , Metabolism , Polymerase Chain Reaction , Promoter Regions, Genetic , Recombinant ProteinsABSTRACT
AIM: To analyze T cell clonality in patients with T cell acute lymphoblastic leukemia (T-ALL). METHODS: The complementarity determining region 3 (CDR3) size of 24 T cell antigen receptor variable ? (TCR V?) region gene was analyzed in peripheral blood mononuclear cell (PBMC) samples from 6 T-ALL cases and 10 normal individuals by using reverse transcriptase-polymerase chain reaction (RT-PCR). The PCR products were further studied by genescan and sequencing analysis. RESULTS: Some TCR V? subfamily T cells display mono- or oligoclonal expansions in 3 cases of T-ALL, predominantly in V?2, V?3, V?6, V?9, V?21 or V?24, respectively. Polyclonal expansions of T cells were found in the other three cases, which could also be found in normal samples. CONCLUSION: A part of T cell acute lymphoblastic leukemia cells may arise from a clonal expansion of TCR V? subfamily T cell. This method may be useful for the detection of minimal residual disease in clinical study of the disease.
ABSTRACT
The TCR Vbeta 24 subfamily genes were amplified in peripheral blood and bone marrow mononuclear cells from 5 cases with acute monocytic leukemia (AML-M(5)) using RT-PCR, to observe the distribution of TCR Vbeta subfamilies. The results indicated that 1 - 19 Vbeta subfamily T cells could be identified in different samples from AML-M(5) cases. The variation of distribution of TCR Vbeta subfamily T cells could be found in different individual samples. The results provided the feature of cell immune function change in skewed distribution of TCR Vbeta subfamily T cells from peripheral blood and bone marrow of patients with AML-M(5).
ABSTRACT
AIM: To analyze T cell clonality in patients with T cell acute lymphoblastic leukemia (T-ALL). METHODS: The complementarity determining region 3 (CDR3) size of 24 T cell antigen receptor variable β (TCR Vβ) region gene was analyzed in peripheral blood mononuclear cell (PBMC) samples from 6 T-ALL cases and 10 normal individuals by using reverse transcriptase-polymerase chain reaction (RT-PCR). The PCR products were further studied by genescan and sequencing analysis. RESULTS: Some TCR Vβ subfamily T cells display mono- or oligoclonal expansions in 3 cases of T-ALL, predominantly in Vβ2, Vβ3, Vβ6, Vβ9, Vβ21 or Vβ24, respectively. Polyclonal expansions of T cells were found in the other three cases, which could also be found in normal samples. CONCLUSION: A part of T cell acute lymphoblastic leukemia cells may arise from a clonal expansion of TCR Vβ subfamily T cell. This method may be useful for the detection of minimal residual disease in clinical study of the disease.