Chikungunya virus infectious clone with capsid protein gene deletion, construction method thereof and application of infectious clone in preparing attenuated vaccine
A technology for chikungunya virus and capsid protein gene, which can be applied in the directions of inactivation/attenuation, virus, antiviral agent, etc., can solve the problems of difficult preservation and virulence recovery, and achieve improved safety and good application. Prospect, effect of good genetic stability
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Embodiment 1
[0055] Construction of an infectious clone of chikungunya virus lacking capsid protein gene and rescued virus:
[0056] This example explores the properties of the infectious clones constructed by exploring the different regions of the missing capsid protein:
[0057] 1. Construction of infectious clones with deletion of capsid protein RNA binding domain (CHIKV-ΔC-115aa) and deletion of all capsid protein genes (CHIKV-ΔC): Synthesize six primers according to CHIKV-WT sequence (GenBank accession no.KC488650) , whose sequences are shown in Table 1 as F1 and R1; F2 and R2; F3 and R3.
[0058] The CHIKV-ΔC-115aa fragment uses CHIKV-WT as a template, uses F1 and R2, F2 and R1 as primers, respectively, and uses PrimeSTAR HS enzyme (purchased from Takara Company) for PCR amplification, recovers the PCR product, and uses the above-mentioned 2-stage PCR The recovered product is used as a template for fusion PCR amplification, the primers are F1 and R1, and the PCR product is recovered...
Embodiment 2
[0071] Comparison of CHIKV-ΔC virus provided by the present invention with CHIKV-WT plaque morphology, growth curve and number of infectious virus particles:
[0072] 1. The CHIKV-ΔC virus rescued in Example 1 and CHIKV-WT were subjected to plaque assay to determine virus titer and morphological comparison: inoculate 1×10 in each well of a 24-well cell culture plate. 5 For BHK-21 cells, when the cell confluency reaches 90%, discard the culture medium in the well, add 100 μl of the virus collected in the above step 3 diluted 10 times with DMEM medium containing 2% FBS, and adsorb at 37°C for 1 h, every Shake well every 15 minutes. After the adsorption was completed, the virus liquid in each well was discarded, and the cover of DMEM medium containing 2% FBS and 2% methylcellulose was added, and cultured in an incubator at 37°C and 5% CO2 for 3 days. After formation, stain with a staining solution containing 1% crystal violet and 3.7% formaldehyde, treat at room temperature for ...
Embodiment 3
[0076] The CHIKV-ΔC virus provided by the invention has genetic stability:
[0077] 1. The CHIKV-ΔC virus (P0 generation) rescued in Example 1 was used to infect BHK-21 cells at a multiplicity of infection of 0.01. After 5 days, the CPE obviously collected the cell supernatant (P1 generation), and each generation passed three strains of virus in parallel. This step was repeated 5 times; the collected cells and supernatants of the P0 and P5 generations were respectively extracted from the viral RNA according to the instructions of the Trizol and QIAamp Viral RNA Mini Kit kits, and stored at -80°C for later use. The PrimeScript One Step RT-PCR Kit was used for RT-PCR detection, and the primers were CHIKV-6791-F:5'-gatgattcacttgcgcttac-3' and CHIKV-8498-R:5'-gatgcttgtagcagctgat-3'.
[0078] 2. Get the supernatant virus liquid that P0, P5 generations collect and carry out plaque morphology comparison (method is the same as embodiment 2); The two are carried out growth curve compar...
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