1, Genetically Engineered Proteins HCD80 / Anti-CD19 ScFv In The Test And In Vitro Functional Studies 2, De-ubiquitination Enzymes For NPM1c And AML1-ETO Ubiquitin Regulatory Mechanisms Preliminary Exploration

In this study, we have succesfully constructed pET32a-hCD80/anti-CD19scFv vector to produce a N-terminal thioredoxin and His tagged hCD80/anti-CD19scFv fusion protein, and increased the yield of soluble hCD80/anti-CD19scFv product in the Rosetta-gami(DE3) strain. Following cleavage of the thioredoxin and His tagged protein with Enterokinase, Enterokinase is removed and non tagged hCD80anti-CD19fusion proteins were purified. ELISA assays showed that the purified proteins could specifically and efficiently bind to the CD19+leukemic cells and elicit specific autologous T cell stimulation. Human primary leukemic B cells decorated with hCD80/anti-CD19scFv were capable of inducing autologous cytolytic activity in vitro. High cell density culture was carried out to produce soluble hCD80/anti-CD19scFv. Pharmacokinetics and distribution of hCD80/anti-CD19scFv in KM mice were studied by125I isotope tracing method. hCD80/anti-CD19scFv fitted to two-compartment model after administration via vein, distribution time was (19.2±10) min, plasma elimination half-life was (11.6±0.7)h, it was mainly distributed in blood, liver, spleen, lung, kidney and excreted via urine. This hold promises for the future study. Several hematopoietic regulators and leukemia specific onco-proteins such as NPMl and AML1-ETO were found to be ubiquitinated by E3ubiquitin ligase and degraded by ubiquitin-proteasome pathway. However, the DUBs that regulate the reverse progress were not reported. To identify the deubiquitinating enzymes (DUBs) that would regulate deubiquitination of NPM1cA and AML1-ETO, We developed a dual fluorescence reportor system coupling FCS technology with knock down of DUBs gene family. The dual fluorescence report vector pCMV-DsRed-IRES-EGFP/NPM1, pCMV-DsRed-IRES-EGFP/NPM1cA and pCMV-DsRed-IRES-EGFP/AML1-ETO were constructed. All the three proteins showed to be very stable proteins in293T cells using this reportor system. We also showed that cytoplasmic localization of NPM1cA is Less stable than the wild type NPM1which is localized in the nucleolus. So the cell-based screen platform showed to be effective and feasible.