Construction Of Genetic Engineering Bacteria Of Recombinant Human Cardiac Troponin Ⅰ

Troponin is the contractile regulatory protein of striated muscle,its complex iscomprised of three distinct polypeptides that are involved in calciumregulation: troponin C, troponin I and troponin C.Troponin I is the subunit that inhibitsactomyosin ATPase activity. The cardiac isform of troponin I is not expressed in anytype of skeletal muscle and is tissue-specific for the myocardium,making it anexcellent biochemical marker for detection of myocardial injury.Studies of patientswith AMI have demonstrated early release of troponin I into the blood stream after theonset of chest pain, reaching peak concentrations at 14-36 hours. The levels remainelvated for 3-7 days after infarction. Measurement of troponin levels thereforeprovides a sensitive and specific determination of myocardial injury over a wide timewindow. The further study provided data supporting a quantitative relationshipbetween the troponin I level and the risk of death in actute coronary syndromepatients. Addtional work has demonstrated troponin I level also increase in non-fatalmyocardial infarction and congestive heart failure.Today,cadiac troponin I has becomea "golden standard" in diagnostic of myocardial infarction.The purpose of this research is that Construction of genetic engineering bacteria ofrecombinant human cardiac troponin I with high stability and expression,in order toobtain recombinant human cTnI protein as materials used in clinical diagnosis andprognosis of myocardial injury and contribute to the research of cTnI diagnosisstandardization.In order to achieve target protein at high levels,two bases changes wererequired,in the second and fourth codons of cDNA sequence.The codonschanges, (Ala2)GCG→GCC and (Gly4)GGG→GGT, do not alter the codingpotential of the DNA.In order to subclone the cardiac troponinⅠgene into the expression vectorpET21a(+).PCR was used to introduced suitable endonuclease restriction sites at otherends of the two genes; NdeI site at the 5′end, the ATG being the first codon of thegene, and BamHI site at the 3′end. The technique also served to amplify the cDNAfor cloning purposes. The Full-length of human cardiac troponinⅠgene was amplified from humancardiac total RNA by RT—PCR and inserted it into a pMD 19—T vector to construct aprokaryotic cloning plasmid. The new plasmid was diagested by restriction enzymeand its sequence was analysed. The cTnI fragment was inserted into pET-21a(+) vectorforming an expression plasmid,then transformed it into BL21 (DE3) bacteria.Proteinexpression was induced by IPTG. The specificity of the expression target protein wasanalysed by SDS-PAGE and RAMP(R) Clinical Reader. In order to detecting thestability, genetic engineering bacteria were continuously passaged.The full-length of human cTnI gene was successfully cloned and the production ofrecombinant human cTnI can reach up to 38％of the total cell protein.The characterof recombinant antigen was good and the genetic engineering bacteria was verystable.Conclusion, successfully constructing genetic engineering bactria of recombinanthuman cardiac troponin with high stability and expression.