Method for making chitosan nanoparticle-pcDNA3.1-hVEGF gene complex
A chitosan nanoparticle, -pcdna3.1-hvegf technology, applied in the field of biomedical engineering, can solve problems such as easy degradation, difficult material compounding, and protein variability
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Embodiment 1
[0007] Embodiment 1 Construction of recombinant plasmid pcDNA3.1-hVEGF
[0008] Plasmid pcDNA3.1 is a eukaryotic expression vector, which constructs a human cytomegalovirus promoter, which can express and regulate the inserted foreign gene. The plasmid itself carries the gene coding sequence of hVEGF, and can express and produce hVEGF under the action of its own expression control elements. There are 17 restriction restriction sites on the multiple cloning site of the plasmid. The BamHI and KpnI regions can be cut with restriction endonucleases, and then T4DNA ligase is used to connect the exogenous gene that has also been double-digested with BamHI and KpnI, so that it is placed under the control of the CMV promoter of human cytomegalovirus, In this way, the eukaryotic expression vector of the foreign gene is constructed (see accompanying drawing).
Embodiment 2
[0009] The preparation of embodiment 2 chitosan nanoparticles
[0010] Adjust the chitosan solution with a certain concentration to pH5.5 with 1N HCl, and take 200ul and a certain concentration of Na 2 Add 200 ul of SO4 solution into the chitosan solution, and quickly mix with a vortex mixer for 30 seconds to obtain the chitosan nanoparticle suspension.
[0011] Determination of morphology, particle size and particle size distribution: the purpose is to investigate the influence of various factors on the preparation process. Take a small amount of nanoparticle suspension and drop it on the copper grid covered with carbon film, let it stand for 2 minutes, blot the suspension with filter paper, then add 2% phosphotungstic acid for negative staining for 2 minutes, and observe the nanoparticle under a transmission electron microscope. granular form. The transmission electron microscope photos are measured with a scale and the particle size is calculated according to the magnific...
Embodiment 3
[0014] Embodiment 3 chitosan nanoparticles-pcDNA 3.1 -Construction of hVEGF gene complex
[0015] Prepared by coacervation method (all the operations are carried out under sterile conditions): first prepare 0.03% chitosan (Sigma) solution, adjust the pH value to 5.5 with 1N HCl, and filter to sterilize with a 0.22 μm filter membrane. with 75mM Na 2 SO 4 (Analytical grade, Beijing Chemical Plant) solution (0.22μm membrane filtration sterilization) to dissolve pcDNA 3.1 -hVEGF recombinant plasmid, so that the final concentration is 200 μg / ml. Take 200 μl of 0.03% chitosan solution and 200 μl of 75 mM Na 2 SO 4 The solutions were placed in an aqueous solution at 55° C. for 20 minutes at a constant temperature. Accurately pipette 200μl of Na 2 SO 4 The solution was added to the chitosan solution and quickly mixed on a vortex mixer for 30 seconds to make chitosan nanoparticles-pcDNA 3.1 -hVEGF suspension, lyophilized into nanoparticles at low temperature, and stored at 4°C...
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