Construction And Prokarytion Expression Of Recombinat Immunotoxin IL6(T22)PE38 Gene

Immunotoxins are target proteins toxin that contain a toxin along with an antibody or growth factor that binds specifically to target cells and kills target cells by enzymatically inhibiting protein synthesis. For the immunotoxin to work, it must bind specifically to and be internalized by the target cells, Some antibodyand immunologic proteins that are smaller than monoclonal antibodies (MAbs), like growth factors and cytokines, have also been conjugated to a protein toxin devoid of its natural binding domain by gene recombination, these binding ligand can carry toxin protein to target cell surface, then kill target cell. In the past ten years, a wide variety of recombinant immunotoxins have been tested against a variety of malignancies in cell culture, in animal models, and in bench.We constructed recombinant immunotoxin on the basis of hIL6 receptor overexpression on some malignancy cell surface, like on the human myeloma U266 and on EB virus-transformed CESS cells, normal cell lack this receptor. Binding of hIL6 ligand can be a vector that carried protein toxin of PE38 to kill cancer cells via binding to a surface IL6R. Recombinant immunotoxin of IL6(T22)PE38 gene was constructed and expressed in Prokaryotic expression system, Killing activity of IL6(T22)PE38 against tumor cell was initial identified in cell culture.1. Construction and Secretory expression of recombinant immunotoxin IL6(T22)PE38 in Bacillus megaterium. Recombinant immunotoxin of IL6(T22)PE38 gene was constructed by two-step overlapping PCR, then insert into KpnⅠand NarⅠmultiple clone site of vector PHis1525. Identify the constructed recombinant plasmid by restriction analysis and transform to WH320 strains of Bacillus megaterium for secretory expression, via the cell wall regeneration, resistance selection. Recombinant immunotoxin was successfully expression of a small amount of secretion in the culture medium supernatant under induction of xylose, but did not showed any cytotoxic activity to SP2/0 myeloma cells. In a word, the secretory expressed recombinant protein IL6(T22)PE38 in medium supernatant of Bacillus megaterium showed no cytotoxic activity.2. High expression of recombinant immunotoxin IL6(T22)PE38 in E.coli and primary cytotoxicity. Amplify recombinant immunotoxin of IL6(T22)PE38 gene from recombinant plasmid of Bacillus megaterium by PCR, then insert into Nco I and Xho I multiple clone site of vector pET28a, Identify the constructed recombinant plasmid by restriction analysis and respectively transform to E.coli BL21 Rosetta blue and pLysS for expression under induction of IPDG, Purify expressed protein. The expressed product was identify by SDS-PAGE and TLC sanning analysis and determined for cytotoxicity by MTS assay. Recombinant proteins in E.coli showed prat of soluble expression. The Rosetta blue expression levels was the highest and the maximum quantitu was 15.3 % in total solublebacterial protein content. Three kinds of recoombinant E.coli produced recombinant toxin cytotoxic activity of all there is and specificity kill human myeloma cells U266 and SP2/0 in vitro. Meanwhile, the three kinds of recombinant bacteria induced conditions were initially optimized. Best conditions induced as follows: IPTG to a final concentration of 1 mmol/L, 28℃induced by 5 hours. The recombinant immunotoxin in BL21(DE3)pLysS expression levels was the highest and the maximum quantity was 18.2 % in total solublebacterial protein content, LD50 of IL6(T22)PE38 cytotoxic activity to U266 cell was 8-15 ng/mL. The successful construction and expression of recombinant toxin IL6(T22)PE38 has laid a foundation for further study.