ABSTRACT
To obtain chicken CD40L protein, the cDNA was prepared from chicken splenic cells and used as a template to clone and amplify CD40L by PCR. The target gene was cloned into pFastBac vector to construct a pFastBac-chCD40L donor plasmid. Recombinant plasmid was transformed into DH10Bac and recombinant Bacmid-chCD40L was obtained. The Bacmid-chCD40L plasmid was transfected into sf9 insect cells to obtain His-chCD40L protein. In addition, the target gene was cloned into pQM01 vector to construct a pQM01-chCD40L plasmid, recombinant plasmid was transfected into HEK 293T cells to obtain Strep-chCD40L protein. The chCD40L protein was purified by affinity chromatography, and the concentration of purified chCD40L protein was determined to be 0.01 mg/mL. Primary cells were isolated from the bursal tissue of 3-week old SPF chickens, and the chCD40L protein was added to the culture medium to stimulate cells. The chCD40L could bind to CD40 on B cells as examined by Western blotting, indirect immunofluorescence assay and flow cytometry, suggesting that chCD40L protein is biologically active. We successfully obtained chicken CD40L protein of biological activity, which laid the foundation in the in vitro culture of primary B lymphocytes for the isolation and diagnosis of virulent IBDV.
Subject(s)
Animals , Baculoviridae/genetics , CD40 Ligand/genetics , Chickens , Cloning, Molecular , Genetic Vectors/genetics , Recombinant Proteins/geneticsABSTRACT
The colorimetric method has been well developed to detect the sensitivity of most antiproliferative drugs with the advantages of objectivity and accuracy. Many parameters including the dosage and the absorbance of crystalline violet, the extraction time of the solvent, plate effect and marginal effect were investigated, then the optimized method was applied further to measure the biological activity during the refolding of recombinant human IFN-? inclusion bodies.
ABSTRACT
The responsiveness of SAC-activated human B cells to human rIL-4 remains controversial. We have proved that rIL-4 definitely supports the proliferation of SAC-activated human tonsillar B cells. B cells preactivated with SAC for 24 hr or 72 hr exhibited the same magnitude of response to rIL-4. However, the 24 hr SAC-B cells showed substantially higher DNA synthesis without supply of rIL-4 than did of 72 hr SAC-B cells. The time that rIL-4 promoted proliferation of B cells is short. The growth factor activity of IL-4 for B cells is strongly unraveled in the first 24 hr cultivation and then, becoming weaker with prolonging of culture period. Two-step assay with 24 hr incubation for each step is introduced for the assessment of rIL-4 activity by SAC-B cells. Anti-? beads activated B cells showed better response to rIL-4 in comparison with SAC-B cells. B cells activated by either anti-? or SAC responded well to rIL-2 or rIL-4.