A Study Of Genetic Engineering On Several Targeting Fusion Protein Drugs

Activated Interleukin 2 (IL2) receptor-positive lymphocytes have been found to accumulate as cellular infiltrates in many diseases, such as autoimmune thyroid disease, type I diabetes, allograft rejection, rheumatoid arthritis, etc. The abnormal T-lymphocytes of adult T-cell leukemia (ATL) and AIDS patients also express aberrant IL2 receptors. It is considered that IL-2 receptor-positive lymphocytes play a crucial role in the pathogenesis of such kind of diseases. Therefore, DL-2 receptor-bearing cells are important targets for immunosuppression. It leads to a therapeutical idea on these autoimmune diseases. So, many kind of fusion immutoxin targeting to IL-2 receptor bvearing cells were constructed. However there is an obstacle for using such kind of fusion proteins as a drug in clinical treatment. That is the immunogenicity of the foreign protein in vivo.The research was divided into four parts.Part I: Construction and induced expression of the fusion protein genes :IL-2(60)-PEZN, IL-2(60)-PEZNK, IL-2-PEZNM, IL-2-PEZNKM, IL-2-K-PEZNM.Part II: Rapid purification of the fusion proteins, bioactivity assay in vitro andpreparation of the antisera against chimeric proteins.Part III: Purification of the fusion protein IL-2-PEZNKM and Fundamentalinvestigation on pharmacological activities.Part IV: Purification of a new fusion protein IL-2-TNFαM and Fundamentalinvestigation on pharmacological activities.In the research of part I: The fusion genes of IL-2(60)-PEZN, IL-2(60)-PEZNK, IL-2-PEZNM, IL-2-PEZNKM, EL-2-K-PEZNM obtained by polymerase chain reaction and deletion mutagenesis were cloned into expression vector. The fusion genes of IL-2(60)-PEZN, IL-2(60)-PEZNK were cloned into plasmid pT7-7 under the T7 promoter and were expressed in E.coli K38 at high level respectively under thermal induction after co-tansformation with plasmid pGP1-2. Desitometric scanning on SDS-PAGE gel revealed that IL-2(60)-PEZN protein, IL-2(60)-...