Viral myocarditis gene vaccine, its preparation method and application
A technology for viral myocarditis and gene vaccine, applied in the field of immunology, can solve the problems such as the inability of gene vaccine to exert immune efficacy effectively, and the difficulty of lasting retention, removal or degradation of naked DNA.
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Embodiment 1
[0023] Example 1. Construction of pcDNA3-VP1
[0024] 1) First carry out the culture of Hela cells and the culture and passage of CVB3 virus. The method is as follows: Hela cells of the human cervical cancer cell line are cultured according to the conventional method, with 10% NBS, 2mM L-glutamine, 100U / ml penicillin and sulfuric acid Kanamycin RPMI-1640 medium at 37℃, 6% CO 2 Cultivation under the conditions, passage once every other day, and gently digest the cells with 0.02% EDTA digestion solution at 37°C during passage, and pass through 1:2 to 1:4 after evenly pipetting. Infect about 5×10 with 100 1 CVB3 virus cryopreservation solution 6 For Hela cells, 80% of the cells were lysed by the replicated virus after 40 hours of culture, the liquid and cell debris were centrifuged at 3000 rpm / min for 20 minutes, and the supernatant obtained was a fresh CVB3 suspension.
[0025] 2) Obtain the CVB3 VP1 gene by RT-PCR from freshly cultured CVB3. The method is as follows: According to t...
Embodiment 2
[0030] Example 2. Preparation of a novel viral myocarditis gene vaccine chitosan-pcDNA3-VP1
[0031] 1) Prepare chitosan solution and pcDNA3-VP1 solution separately, the method is as follows: First, prepare 0.02% chitosan solution with pH 5.7, weigh 0.02g chitosan (Sigma, MW=39000), first use 500μl~1ml 1% HAc solution to It dissolves slowly and is placed at 37°C for 1 hr. Then weigh out 0.042g NaAc, and use 80ml H 2 O dissolves, add the above completely dissolved chitosan solution, and after mixing, adjust the pH to 5.7 with 1N NaOH, which is a 0.02% chitosan solution with pH 5.7. PcDNA3 plasmid with 50mM Na 2 SO 4 Dissolve, DNA concentration is 1mg / ml, store at -20℃ for later use.
[0032] 2) The chitosan-pcDNA3-VP1 complex was prepared by the complex co-precipitation method. The method is as follows: In a 55°C water bath, drop a 0.02%, pH 5.7 chitosan solution into a 80ug / ml plasmid DNA solution at a high speed. Shake for 20 seconds to form a uniform chitosan-pcDNA3-VP1 complex ...
Embodiment 3
[0034] Example 3. In vitro expression of a novel viral myocarditis gene vaccine chitosan-pcDNA3-VP1
[0035] Add 100ul of chitosan-DNA solution containing 1μg pcDNA3-VP1 DNA directly to 2×10 5 On the surface of Hela cells, add 2μl Lipofectamine with 1μg pcDNA3-VP1 DNA TM -2000 transfection complex as a control (1-3×10 5 A number of cells are seeded into a 24-well plate, and 2ml of fresh antibiotic-free culture medium is replaced in the first 4 hours, and the cell density is 90-95%. Take 2ul FuGENE6 TM Add the eukaryotic cell transfection reagent to 45μl of serum-free 1640, mix carefully, at room temperature for 5 minutes, while taking 1μg of plasmid pcDNA3-VP1DNA purified by QIAGEN purification kit into 50μl of serum-free 1640, carefully mix, room temperature for 5min, 50μl FuGENE6 TM Eukaryotic cell transfection reagent was added dropwise to 50μl DNA, gently pat well, 20min at room temperature to promote the formation of complexes; Wash Hela cells 3 times with serum-free 1640, ad...
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