Gene Cloning, Expression And Purification Of Human FLT3 Ligand

FLT3 ligand(FL) was discovered by Lyman et al. They used a soluble form of the FLT3 receptor to clone the gene of FL from murine and human cell lines. It consists of 235 amino acids and consists of a 26 amino acid signal peptide, a 156 amino acid extracellular domain, a 23 amino acid transmembrane domain and a 30 amino acid cytoplasmic tail. The FL is widely expressed in both murine and human tissues. Highly level of FL mRNA on human tissue Northern Blot has been reported in peripheral blood mononuclear cells. Serum levels of FL are highly elevated in patients with hematopoietic disorders such as acquired aplastic anemia and myelosuppression. FL is one of credible items that evaluate the hematogenic function. FL is a cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor/stem cells .FL, administered either alone or in combination with other cytokines, can stimulate the proliferation of progenitor/stem cells. FL stimulates the progenitor/stem cells into cell cycle and it can be used to in gene therapy as it mobilized progenitor/stem cells might be excellent targets in gene therapy. The researches demonstrate the antitumor activity of FL, because it induces expansion of functional DCs, NKcells and TCL cells in vivo. It also has antivirus and antibacterium activity. The newly research demonstrates the profound radioprotective effects of FL.In order to get the FL in quantity and study the biological function. Firstly mononuclear cells were gained from the human peripheral blood by the Ficoll's centrifugation. A cDNA encoding soluble FL was cloned through RT-nested PCRfrom the total RNA extracted from human peripheral blood mononuclear cells and identified by analyzing the nucleotide sequences. The human FL gene was subclone into expressing vector pRSET-B. The recombinant plasmid pRSET-B-FL was transformed to BL21(DE3), then FLT3 ligand was expressed under the inducement of IPTG. The fused protein was purified by ion exchange methods of SP-Sepharose Fast Flow and Q-Sepharose Fast Flow. SDS-PAGE and Western-blot was employed to identify the expression of human FLT3 ligand. FL gene with a length of 468 bp was isolated from human peripheral blood mononuclear cells. Sequence analysis revealed the sequence of FL gene was consistent with relevant reports. FL gene was cloned into expressing vector pRSET-B identified by enzyme digestion of EcoRl and Hind III enzyme. SDS-PAGE showed that a Mr 24 000 fused protein was expressed in E.coli. The purity of the fused protein obtained by ion exchange methods was 90%. Wenstem-blot showed the fused protein could be recognized by anti-FL antibody.FL gene was obtained by RT-PCR amplification. The expressing vector of FL was successfully constructed and expressed in E.coli. The fused protein is purified preliminarily. The study will provide necessary conditions for obtaining rhFL protein in large scale.