Double-stranded RNA compound AUK and application thereof in vaccine preparation
A compound and vaccine technology, applied in the direction of antisense single-stranded RNA virus, vaccine, veterinary vaccine, etc., can solve the problems of limited application, toxic side effects, unstable chemical structure, etc.
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Embodiment 1
[0061] Embodiment 1. Preparation of complex AUK
[0062]Weigh Poly A 0.1132g, add it to 49ml PBS (10mM, pH7.2), dissolve it as Poly A stock solution; weigh Poly U 0.1152g, add it into 49ml PBS (10mM, pH7.2), dissolve it as Poly U stock solution; prepare 80mg / ml kanamycin sulfate stock solution with PBS (10mM, pH7.2). Heat the prepared Poly A stock solution, Poly U stock solution, and Kanamycin Sulfate stock solution in a water bath to 50°C, and mix the three stock solutions to make the Poly A: Poly U: Kanamycin Sulfate stock solution in the mixture The liquid volume ratio is 0.49:0.49:0.02, and each 1ml of the preparation obtained contains Poly A (1.02mg), Poly U (0.98mg), and kanamycin sulfate (1.6mg). The mixture was incubated in a water bath at 50°C for 30 minutes, then incubated at 0°C for 1 hour, and finally filtered and sterilized using a 0.22 μm sterile filter to obtain the complex, which was named AUK. The concentration of the obtained AUK solution was 2 mg / ml (kanam...
Embodiment 2
[0063] Example 2. Property detection of complex AUK
[0064] 1. Molecular weight detection
[0065] The molecular weight of the compounds was analyzed using 1.5% agarose gel electrophoresis. RNA loading buffer (Thermo Fisher), DL2000 molecular marker (DL2000 Marker) (0.2-2KB, Takara) was used to estimate the sample size distribution range. The results of agarose gel electrophoresis figure 1 As shown, 1 is Poly I:C solution (2mg / ml), 2 is Poly A:U solution (2mg / ml), 3 is AUK solution (2mg / ml). The length distribution of complex AUK is between 500-1200bp, and the molecular weight range is between 340-816KDa. The length and molecular weight of Poly A:U and Poly I:C are close to AUK.
[0066] 2. Ultraviolet absorption spectrum detection
[0067] Microplate reader (Biotek Synergy TM H1) Scan the spectrum of the complex AUK in the range of 230nm-300nm to detect its ultraviolet absorption spectrum. The result is as figure 2 As shown, the UV absorption peak of the complex AUK...
Embodiment 3
[0094] Example 3. Vaccine Preparation and Immunization
[0095] 1. Preparation of virus liquid
[0096] H9N2 virus was used as antigen to evaluate the effect of complex AUK in chicken immunization experiments. Prepare the H9N2 virus solution as follows:
[0097] (1) Get the H9N2 subtype avian influenza virus and make appropriate dilution (such as 10 h) with sterilized saline -4 or 10 -5 diluted), inoculate 0.1ml into the allantoic cavity of each embryo, seal the pinhole, and continue to incubate at 36-37°C without turning the eggs.
[0098] (2) Within 48 hours after inoculation, the eggs were illuminated once a day, and the chicken embryos that died within 48 hours were discarded. After that, take photos every 4 to 6 hours, and take out the dead chicken embryos at any time until 96 hours. After 96 hours, regardless of whether the chicken embryos are dead or not, all the chicken embryos are taken out, the air chamber is upright, and cooled at 2-8°C for 4-24 hours.
[0099] ...
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