The present study was designed to fused enzymatically cytotoxic DAB(389) (containing the N-terminal 388 residues of diphtheria toxin) to alpha melanocyte-stimulating hormone (alpha MSH) for constructing DAB(389)(Gly(4)Ser)(2)-alpha MSH immunotoxin. In experiments, DAB(389) (Gly(4)Ser)(2)-alpha MSH fragments were amplified from pET28a-DAB(389)(Gly(4)Ser)(2)-mIL3 vector by PCR with both the upstream primer containing His.Tag and DAB(389) sequences and the downstream primer containing full alpha MSH sequence and (Gly(4)Ser)(2) complementary sequences. DAB389(Gly(4)Ser)(2)-alpha MSH fragments were cloned into a prokaryotic expression vector pET28a(+), forming pET28a-DAB(389) (Gly(4)Ser)(2)-alpha MSH. Immunotoxins were expressed in optimized BL21 E. coli system under IPTG induction, followed by purification. Immunotoxins targeted to A549 lung adenocarcinoma cells which bear alpha MSH receptor. By receptor-mediated internalization, the immunotoxin was specifically cytotoxic to A549 cells (IC50 = 3.13 mu g/ml) versus BGC-823 cell control and significantly inhibited the growth of A549 solid tumors carried on nu/nu mice.
[1]Jilin Univ, China Japan Union Hosp, Dept Thorac Surg, Changchun 133033, Peoples R China.
[2]Jilin Univ, China Japan Union Hosp, Dept Orthoped, Changchun 133033, Peoples R China.
[3]Jilin Univ, China Japan Union Hosp, Dept Endoscopy, Changchun 133033, Peoples R China.
[4]Jilin Univ, Bethune Hosp 1, Dept Pediat, Changchun 130021, Peoples R China.
[5]Hebei Univ Engn, Coll Med, Handan 056009, Peoples R China.
(8.0+极速模式)