Mitochondrial positioning polypeptide, positioning system and application thereof
A positioning system and mitochondrial technology, applied in the field of biomedical basic research, can solve the problems of poor effect, easy quenching of antibody and dye fluorescence, unsatisfactory color rendering effect, etc., to achieve no toxic side effects, efficient targeted drug delivery, The effect of wide application value
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Embodiment 1
[0091] Example 1 Screening of Mitochondrial Localized Protein Sequences
[0092] Analyze the sequence information of mitochondria-related proteins and screen the mitochondrial localization signal sequence, as shown in Table 1:
[0093] Table 1 Mitochondrial localization signal protein sequence
[0094]
Embodiment 2
[0095] Embodiment 2DNA sequence synthesis design
[0096] Using the self-constructed plasmid pcDNA3.1-MCS-EGFP as the carrier, select the upstream Hind III and the downstream EcoR I restriction sites, and ensure that the inserted sequence and EGFP protein are expressed and translated into a fusion protein without frame shift mutation or premature termination During translation, a base "G" is added to the end of each mitochondrial localization signal. The gene DNA sequences of the three mitochondrial localization signals are shown in Table 2:
[0097] Table 2 Mitochondrial localization signal DNA sequence
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[0099]
Embodiment 3
[0100] Embodiment 3 constructs fusion expression plasmid
[0101] Sequencing alignment of transfer expression vectors and plasmids. The three mitochondrial localization signal DNAs obtained in Example 2 were digested by HindIII and EcoRI in a water bath at 37°C for 2 hours, and then the target fragments were recovered by 1% agarose gel electrophoresis again. Similarly pcDNA3.1(+)-MCS-EGFP empty plasmid ( figure 1 ) was also digested by HindⅢ and EcoRI in a water bath at 37°C for 2 hours, and the fragment after double digestion was ligated with the vector by T4 DNA ligase overnight at 16°C. Transfer the ligated fragments to DH5α-competent Escherichia coli, ice-bath for 5 minutes, and immediately ice-bath for 5 minutes at 42°C for 1 minute, add 600 μL of blank LB medium and shake the bacteria at 37°C for 1 hour, centrifuge at 1200 rpm for 3 minutes, discard the supernatant, Keep 20 μL and blow it evenly, spread it on the ampicillin-resistant LB culture plate, and incubate over...
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