Mucosa adjuvant based on deoxyribose nucleic acid (DNA) receptor as well as preparation and application thereof
A mucosal adjuvant and sensor technology, applied in the field of genetic engineering, can solve problems such as poor effect, and achieve the effect of promoting activation, effectively resisting CVB3 infection, and enhancing sIgA antibody and T cell response
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Embodiment 1
[0058] Example 1 Construction and in vitro expression of pcDNA3.1-pVP1 target antigen gene vaccine
[0059] 1) CVB3VP1 gene was obtained from freshly cultured CVB3 by RT-PCR. According to the VP1 cDNA sequence of CVB3Nancy strain reported in the literature (Klump WM, et al. Complete nucleotide sequence of infectious Coxsackievirus B3 cDNA J Virol, 1990, 64(4): 1573), the upstream and downstream primers of VP1 were designed with Hind III and BamH I restriction site: upstream: 5′-CCCAAGCTTGCCACCATGGGCCCAGTGGAAGACGCG-3′; downstream: 5′-CGGGATCCTTACTAAAATGCGCCCGTATTTGTC-3′. The liquid phase viral RNA was extracted according to the RNAex Reagent&System Kit of Shanghai Huashun Biological Company. The cDNA was obtained by reverse transcription using the 2-N First Strand cDNA Synthesis Kit of Shanghai Sangon Biotechnology Co., Ltd. Then specific PCR was carried out with VP1 primers, and the product was VP1 gene with a length of 852bp (as shown in SEQ ID NO: 7). DNA sequencing ident...
Embodiment 2
[0061] Construction and expression of embodiment 2pAIM2 eukaryotic expression vector
[0062]Mouse RAW264.7 cells were cultured, stimulated with a final concentration of 20ng / ml IFN-γ for 24 hours, harvested to extract RNA and reversed to obtain cDNA. AIM2 upstream and downstream primers were designed with Hind III and XhoI restriction sites: upstream: 5′-CCCAAGCTTGCCACCATGGAGAGTGAGTACCGGG-3′; downstream: 5′-CCGCTCGAGTCACTCCACACTTTTCATGTC-3′. Then specific PCR was carried out with AIM2 primers, and the product was the AIM2 gene with a length of 1065bp (as shown in SEQ ID NO: 1). After double-digestion, it was connected to the pcDNA3.1 vector, and the DNA sequencing service center of Invitrogen Company carried out DNA sequencing identification, which confirmed that the construction was correct.
[0063] In vitro expression of pcDNA3.1-AIM2: with Lipofactamine TM -2000 helper pcDNA3.1-AIM2 plasmid to transfect 293T cells: 2 μl Lipofectamine TM Mix with 45μl 1640 for 5 minutes...
Embodiment 3
[0064] Example 3 Preparation of oligo-chitosan-pAIM2 mucosal adjuvant nanoparticles
[0065] 1) Large-scale preparation of plasmid DNA: According to the QIAGEN Plasmid Mega Kit, remove impurities and bacterial endotoxins to obtain purified plasmids.
[0066] 2) Prepare oligo-chitosan solution and pcDNA3.1-AIM2 solution respectively, the method is as follows: first prepare 0.4% oligo-chitosan solution with pH 5.5, weigh 0.4g chitosan oligo-chitosan (MW about 3000-6000, The degree of deacetylation is 75%-85%), slowly dissolve it with 100ml of 10mM PBS, and place it at 37°C for 1h. Filter through a 0.4 μm filter and save for future use. The pcDNA3.1-pAIM2 plasmid was dissolved in 10mM PBS, the DNA concentration was 25μg / ml, and stored at -20°C for future use.
[0067] 3) Prepare oligo-chitosan-DNA nanoparticles by co-precipitation method (co-aggregation method), as follows: in a water bath at 55°C, add 0.4% oligo-chitosan solution with pH 5.5 to 25ug / ml plasmid DNA solution dro...
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