Gene Synthesis, Prokaryotie Expression And Monoclonal Antibody Preparation Of Human Myohaemoglobin Protein

In order to prepare the highly specific and highly sensitive anti-myoglobin monoclonal antibodies whichare used for monitoring clinical cardiovascular and cerebrovascular diseases, two-step method was used in this study for the synthesis of human myoglobin total gene, and prokaryotic expression system was used to express myoglobin (Myo) protein which was purified through affinity purification subsequently. At the same time,the purchased myoglobin was used to immunize mice for preparation the high-affinity monoclonal antibody, and finally a panel of monoclonal antibodies with the most specificity and sandwich pairing were screened using the expressed protein in E. coli. The result are as follows:Expression of the total Myo gene:Through optimizing Myo codons, the codons E. coli usually used were got, and the Myo total sequences was got through reverse transcription, The Myo total gene was synthesized through the two-step method. The construction of Myo expression vector:Myo fragment was linked into the pMD18-T vector to get a recombint Myo-pMD18-T vector. The prokaryotic expression vector of Myo was obtained through linking the digested Myo fragment from Myo-pMD18-T into the prokaryotic expression vector pET-28a.Expression and purification of the recombinant Myo protein:Myo-pET-28a was transformed into competent cells BL21(DE3) to get expressed E.coli strains. By IPTG induction and optimization of the expression conditions, the Myo-His fusion protein was finally got under the best temperature, time and concentration The purification of Myo-His fusion protein was utilized by nickel columns, and the purified protein was acquired after dialysis and concentration. Preparation of monoclonal antibodies:The spleen cells derived from the immuned mice with the purchased myoglobin were fused with the lymphoma cells, and22cell strains secreting specific antibodies were screened. Five high-affinity antibodies were ultimately acquired by screening of sub-cloned secreted cells.Monoclonal antibodies:Through examining purified Myo antigen obtained in the expression experiment by Western Blot, all the screened antibodies can reacted with pure Myo antigen, expcept that#16-3.3reacted weakly, therefore#14-4.3,#17-1.2,#18.2and#19-2.3were high affinite and highly specific monoclonal antibody, among them#17-1.2can be used in sandwich paired experiments with the other three antibody.In summary, the highly sensitive and specified Myo monoclonal antibodies were screened in this study. Especially, these antibodies could be used for the preparation of detection kit in clinical of acute myocardial infarction to lay the foundation for human heart diseases clinic. Additionally, these monoclonal antibodies can be used in sandwich paired experiments, and also useful in antibody chips which will be prepared and developed in future. It provides the possibility to establish a new rapid joint method of diagnosis of cardiac disease.