Application of small lipid-binding peptide ssip1 in mediating its fusion protein into cells
A technology of fusion proteins and small peptides, applied in the field of molecular biology
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Embodiment 1
[0029] Example 1 Acquisition of SSIP1-GFP protein
[0030] 1. Construction of SSIP1-GFP (Green fluorescent protein, green fluorescent protein) prokaryotic expression vector
[0031] Total RNA was extracted from 14-day-old Arabidopsis leaves by Trizol method, and cDNA was synthesized by reverse transcription according to the instructions of TaKaRa PrimeScript RT reagent kit with DNA Eraser kit using 1 μg of RNA as a template. Using Arabidopsis cDNA as a template, design primers to amplify the CDS sequence of SSIP1 by PCR (94°C, 5min; 94°C, 30s; 55°C, 30s; 68°C, 40s (back to step 1: 33 cycles) ; 72°C, 5min, 16°C, forever), upstream primer SSIP1-F: 5'-AAGCTTGATATC GAATTC ATGAGCGGCAAAACATCG-3′ (the underline is the EcoRI restriction site), downstream primer SSIP1-R: 5′-TTATCTAGAACTAGT GGATCC TTATTTCTTCTGTTCGTCATTCTTCT-3' (the underline is the BamHI restriction site), the PCR product was subjected to agarose gel electrophoresis, and the gel was recovered. The empty vector pGre...
Embodiment 2
[0034] Example 2 Liposome-embedded SSIP1-GFP protein into protoplast or cell experiment
[0035] The above-mentioned purified SSIP1-GFP or empty GFP protein induced by prokaryotes was mixed with different solvents (1×PBS, 5mM glucose, 10% glycerol, 25% Liposome 2000, 20% Liposome 2000, 125mM CaCl 2 )mix. Such as figure 2 As shown, it was found that SSIP1-GFP could be completely dissolved in 1×PBS and 5mM glucose, micro-embedded in 10% glycerol, and fully embedded in 25% liposomes (PEG), in 125mM CaCl 2 In 20% PEG, larger lipid droplets were formed.
[0036] At the same time, it was found that SSIP1-GFP could be better embedded in 25% liposome 2000 to form dispersed lipid droplets, while the empty GFP protein could not be better embedded in liposome 2000. Alternatively, in 125mM CaCl 2 In solution, both SSIP1-GFP and empty GFP formed precipitates. Therefore, because the culture solution (W5) of plant protoplasts contains 125mM CaCl 2 Therefore, direct addition of purified ...
Embodiment 3
[0040] Example 3 Experiment of SSIP1-GFP protein entering embryonic cells
[0041] After embedding the SSIP-GFP small peptide (2.5ug / mL) in 25% liposomes, open the unfertilized floral organ (Arabidopsis thaliana flower bud) that has just turned white, and slowly add it dropwise to the stigma. close, such as Figure 6 As shown, different degrees of green fluorescence can be seen in the pollen ducts at 24-48h, and green fluorescence can also be observed in the fruit stalk and pericarp of the grown pods, indicating that the SSIP-GFP fusion protein can move in vivo and accumulate to the embryo. It shows that SSIP1 can be used as a peptide guide carrier, which can quickly guide the target protein into embryonic cells, and this method does not involve genome changes.
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