Construction Of Gene Engineering Bacteria Of Recombinant Human Myoglobin And Protein Primary Purification

Myoglobin is a heme-protein similar to hemoglobin, and is a unique pigment-protein in striated muscle. Its relative molecular mass is 17KD, and is about 0.1% to 0.2% of muscle protein total amount. Its molecular structure is similar to subunit of hemoglobin, constitutes of one polypeptide chain and one heme molecule. Human myoglobin polypeptide chain contains 153 aminopeptides, its structure arrangement is extremly similar to the N chain of hemoglobin.Myoglobin used as a chemical mark of myocardial damage has been almost 30 years. At present it has been generally used in early diagnosis of AMI. The impaired cadiocyte is releasing myoglobin into peripherical blood when AMI occurs. About 1 to 2 hours after the invasion, the amount of myoglobin in serum was abnormally step up, after 72 hours the value was cut down to normal level. ECG is one of the reliable diagnostic methods of AMI early diagnosis. The long-time clinical practice has confirmed that if Mb detection is used with ECG, the early diagnosis rate of AMI will be raised up. Some research results has showed that if the ECG examination and Mb detection will carried out on the pectoralgia patients at the same time, the AMI diagnosis rate will be hightened from 62%(ECG single-using) to 82%. But, most of the Mb diagnosis kits in clinical use have high price and come from overseas. All of this restricts the widespread use of Mb in early diagnosis of AMI. So, it is urgent to have our own intellectual property rights of this kit. Extracting the protein from cardiac muscle is the traditional method, but the source of human cardiac muscle is deficient and the quality can't be ensured. All of above make the yield and the activity of the protein low. So we constructed stable, high-expression gene-engineering bacteria of human myoglobin by gene-engineering technique and got the high-purity product.The objective of the research is to construct stable, high-expression gene-engineering bacteria of human recombinant myoglobin, provide diagnostic reagent for diagnosis and prognosis of early myocardial damage, and promote the standardization of cardiac myoglobin diagnosis.In order to subclone the myoglobin gene into the expression vector pET21a(+). PCR was used to introduced suitable endonuclease restriction sites at each ends of the two genes;NdeI site at the 5 ' end,the ATG being the first codon of the gene,and HindIII site at the 3 ' end. The technique also served to amplify the cDNA for cloning purposes. The Full-length of human myoglobin gene was amplified from human cardiac total RNA by RT—PCR and inserted it into a pMD18—T simple vector to construct a prokaryotic cloning plasmid. The new plasmid was diagested by restriction enzyme and its sequence was analysed. The myoglobin fragment was inserted into pET-21a(+) vector forming an expression plasmid, then transformed into BL21 (DE3) bacteria. Protein expression was induced by IPTG. The specificity of the expression target protein was analysed by SDS-PAGE and RAMP(?) Clinical Reader. Purify the recombinant protein by Ammonium sulfate precipitation and dialysis in order to maintain the purity of the antigen for monoclone-antibody preparation.The full-length of human myoglobin gene was successfully cloned and the production of recombinant human myoglobin can reach up to 15% of the total cell protein. The purity of the antigen is 80%, reached the purity for monoclone-antibody preparation.Conclusion: successfully constructing genetic engineering bactria of recombinant human myoglobin with high stability and expression, and primary purifying the recombinant protein.