Human Cardic Troponin I And Preparation Of Monoclonal Antibody Against CTNI

Human cardiac troponin I (cTnI) is an important biochemical marker for myocardial injury and prognosis of myocardial injury. For the sensitive and a wide time window, cardiac troponin I has become a"golden standard" in diagnostic of myocardial infarction. At present, the clinical cTnI diagnostic kits are from abroad or domestic assembly by imported raw materials, which not noly increasing the test cost but also increasing the economic burden of patients. On the other hand, cTnI results varied large sacles among different manufacturers. It is very important to exploit domestic-made and standardized test product of cTnI.However, it is hard to be prepared in quantity. In order to obtain unlimited cTnI proteins, we constructed genetic bacteria pET-11a-cTnI-BL21(DE3) to produce purpose proteins with the genetic engineering technique. The designed recTnI without extra gene segments was native to the nature protein of cTnI. The optimal expressed conditons from the active rhcTnI proteins were determined using response surface methodology (RSM) by Box-Behnken design. The optimal conditons for expressing the rhcTnI proteins cTnI were: induced OD600value of1.0, induced temperature of30.0℃, and induced time of4.5h. The verification result of rhcTnI proteins from total proteins expressing obtained and amounted to32.63%with response surface experiment optimization higher increasd by8.4%.Ion exchange chromatography and Phenyl-Sepharose FF procedures were used to purify cTnI from E.coli. SDS-PAGE and the densitometer scan software shew the purity of the purified purpose proteins amounted to72%. The immunological activity of the purified hcInI was analyzed by western blot assay after SDS-PAGE, and the result indicated that the recombinant protein was well immunologic affinity.The recombinant human cardiac troponin I was successfully expressed at large scale, which will be used in producing monoclonal antibody and contribute to the research of cTnI diagnosis standardization.With the genetic engineering technique, we constructed the genetic bacteria pColdTF-cTnI-BL21(DE3) expressing the cTnI-His. The advantage of recombinant protein expressed in Escherichia coli is easy to grow and purify by Ni-NTA affinity chromatography which takes advantage of the affinity between Ni2+and His. The high purified cTnI-His proteins of98%were coated as the antigen for ELISA.The purified cTnI proteins were used to immune the BALB/c mouse. When the immunizing potency of the mouse was1:50000by the indirect ELISA, the spleen were combined with the SP2/0hybridoma cells to prepare the monoclonal antibody against cTnI.Under subcloning and verifing with the indirect ELISA,4hybridomas, named B2-B4-F12, B2-D2-F6, B2-C2-A6, B2-G4-E7, could serect cTnI McAb. B2-D2-F6was selected to cultivated at large scale and purified by Protein-A affinity chromatography, and the purity was amounted to90%. The titer of polyclonal antibodies was1:5000and well immunologic affinity with the western-blot assay.In conclusion, we obtained the recombinat plasmid pET-11a-cTnI-BL21(DE3) and pColdTF-cTnI-BL21(DE3) expressed the target proteins and the detector proteins His-cTnI. Then, we produced affinity monoclonal antibody and polyclonal antibody against to cTnI, contributed to the research of cTnI diagnosis standardization.