Construction And Expression Of Recombinant Hemoglobin Prokaryotic Coexpression Vector

Transfusion fluids can treat a variety of clinical conditions,and the need for it is continually increasing all over the world.At the same time,donated blood is a limited resources with many side effects.This induced people to search for a whole blood substitute and use the substitute as transfusion fluids to treat the clinical conditions.One such alternative involves the use of acellular recombinant hemoglobin(Hb)as an oxygen carrier,that is to say,the recombinant hemoglobin-based oxygen carrier(rHBOCs).The reseach of rHBOCs has many years of history.On the basis of predecessors,our factory proposes a way to construct a novel rHBOCs.Construct albumin-Hb fusion protein and lead three point mutations into the Hb gene in order to overcome the side effects with transfusion of rHBOCs.Objective: First,the aim of our experiment to construct albumin-Hb fusion protein is to enlarge the Hb molecular weight to treat the nephrotoxicity.On the other hand,in order to decrease the NO affinity of Hb,we introduced three point mutations into Hb gene.This method can deal with the hypertension when transfused with rHBOCs.Afterwards what we need to do is expression the rHBOCs in vitro.Methods and results: Connecting the albumin gene and the Hb?gene with a short linker DNA sequence,at the same time,introducing the three point mutation with overlapping extension PCR method.Afterwards,using specific restriction endonuclease digestion and enzyme linking with the protokaryon coexpression vector pet duet1,and introducing the constructed coexpression vector to the E coil expression strain Origami DE3,using IPTG to induce the expression of target gene.This experiment has successfully constructed a protokaryon coexpression recombinant Hb vector,and has successfully induced the expression of the recombinant Hb gene.This experiment was established for purifying the recombinant Hb and using the purified recombinant Hb for clinical trials in the future.