| Objective:The genes of fusion protein muscle specific receptor tyrosine kinase (MuSK)-mCherry were transfected in human embonic kidney HEK293cells for expression,and furthermore used as antigens in the detection of antibodies against MuSK(MuSKAb)in patients with myasthenia gravis(MG).Methods:E coli DH5a which were transfected with plasmid pMT/BiP/V5-His (MuSK-mCherry)were cultured,and then the plasmid was extracted and put onto the gel imaging system after electrophoresis in order to confirm whether the plasmid solution contains pMT/BiP/V5-His(MusK-mCherry).The humna embryonickidney HEK293cells were cultured to a good condition in6well plate and transfected with plasmid pMT/BiP/V5-His(MusK-mcherry).The cells were collected after24hours’ transfection and sealed wuth the anti-fluorescence mounting mediium.The fluorography was observed in confocal microscope under the condition of excitation wavelength of587and emission wavelength of610nm in order to confirm the expression.The cells were collected every6hours during the48hours after transfection.The protein were extracted from the cells by the method of cell disruption.The proteins were checked in western blot to confirm the expression of different time after transfectionResult:A5647bp band,which is in accordance with the plasmid pMT/BiP/V5-His(MusK-mCherry)was found in gel electrophoresis The HEK293cells showed red fluprescent imaging after transfected with the plasmid pMT/BiP/V5-His(MusK-mCherry).A72.4KDa band,.which is in accordance with the MuSK-mCherry fusion protein was observed in western blot,and a different expression with time observed.Conclusion:The plasmid pMT/BiP/V5-His (MuSK-mCherry) containing MuSK-mCherry fusion protein can be expressed in HEK293cells. The MuSK-mCherry fusion proteins have been obtained and will be used as antigens in the future determination of MuSKAb in MG patients. |
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