Gene Clone, Expression And Purification Of Murine Flt3l As Well As Identification Of Its Biological Activity

Objective: To obtain the cDNA of mouse FMS-like tyrosine kinase 3 ligand and construct the prokaryotic expression vector PGEX-4T3/mFL as well as identify its biologic activity following expression and purification of FL protein in E.coli.Methods: The cDNA encoding FL was cloned by RT-PCR from the C57BL/6 mouse spleen total RNA and identified by analyzing the nucleotide sequences.The mouse FL gene was subcloned into the expression vector PGEX-4T3,and the recombinant plasmid PGEX-4T3/FL was transformed to BL21(DE3) for protein expression under the induction of IPTG.The fused protein was purified by GST affinity chromatograph and detected by SDS-PAGE. Finally, The biological activity of resultant FL protein was identified by examination of its capacity of induction of generation of pDC from bone marrow cells.Results:1,DNA sequence analysis showed that the cDNA encoding FL is exhibit 99% homology with GenBanK reports which accession number is NM₀13520.3. The minor difference is due to a C substitute original T at 688th basic group of cDNA sequence, however, this point mutation does not change the amino acid because it is a degeneracy of code.2,The fusion protein was highly expressed by the prokaryotic expression vector PGEX-4T3/mFL under the inducement of IPTG in the BL21, and SDS-PAGE analysis revealed that the fusion protein had a molecular weight of about 52 000 after GST affinity chromatograph, which is in accordance with the theoretical value.3,In vitro culture for generation the pDC from bone marrow cells with the mFL protein indicated that the resultant mFL exhibited biological activity.Conclusions: The cDNA of mouse FL was cloned and the prokaryotic expression vector PGEX-4T3/mFL was constructed successfully.The expressed fusion protein of mFL exhibited our expected biological activity.This study provided a foundation for further in vitro or in vivo functional experiments as well as preparation of mouse FL protein on a large scale.