Nebulizable tuberculosis vaccine for transnasal administration comprising paramyxovirus vector
A technology for tuberculosis vaccines and paramyxoviruses, applied to viruses/bacteriophages, viruses, and medical raw materials derived from viruses/phages, etc., can solve the problems of inability to fully inhibit adult tuberculosis and low efficacy, and achieve high convenience and efficacy of administration Effect of high and high cost reduction
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Embodiment 1
[0054] Example 1: Construction of the antisense rhPIV2 (M stop) genome with M protein deletion by introducing a stop codon
[0055] figure 1 , shows a summary of the antisense rhPIV2 (M stop) genome recombined with a stop codon at a defined position in the gene encoding the M protein. Represents two types of cDNA recombined into Δ119 (making AAG at position 259 of the M protein gene TAG) and Δ289 (making ATG at position 89 of the M protein gene TAG and making AAG at position 259 TAG) downstream of the T7 promoter as cDNA The case of plasmid vectors of antisense rhPIV2 genomes of M protein-deficient hPIV2 (rhPIV2(Δ119) and rhPIV2(Δ289) respectively) (collectively referred to as rhPIV2(M-terminated)). In constructing hPIV2 (M-terminated) deficient in the M protein, genetic techniques known to those skilled in the art (for example, the PCR method) can be used.
Embodiment 2
[0056] Embodiment 2: the preparation method of rhPIV2 (M termination)
[0057] Next, a method for recovering viral particles from a plasmid containing an antisense rhPIV2 genomic cDNA inserted downstream of the T7 promoter will be described. exist figure 2 A summary of the method is shown in . will be like figure 1 The antisense rhPIV2 genomes (rhPIV2, rhPIV2(Δ289), rhPIV2(Δ119)) constructed by the operations shown above were transfected into cells expressing T7 RNA polymerase (eg, BSR-T7 / 5). At this time, three expression vectors of hPIV2-NP, hPIV2-P, and hPIV2-L, which are vectors expressing hPIV2 polymerase units (ie, NP protein, P protein, and L protein), were transfected together. In addition, methods known to those skilled in the art (Lipofectamine is used in this embodiment) can be used for DNA transfer.
[0058] Co-cultivation with Vero cells was carried out 5 to 7 times every 48 hours, and the cytopathic effect (Cytopathic effect: CPE) was confirmed with an effic...
Embodiment 3
[0061] Example 3: Construction of antisense genomes (ΔM / rhPIV2, ΔF / rhPIV2) with complete deletion of M gene or F gene
[0062] Such as image 3 As shown, one of the Sca I-Kpn I restriction enzyme sites encoding the M gene, the F gene and a part of the HN gene was introduced into the plasmid containing the genomic gene of hPIV2 at the Sca I-Kpn I restriction enzyme site of the pUC118 vector The roughly 5.5 kb gene between them.
[0063] Regarding the construction of the M gene-deficient genome, ΔM / rhPIV2 in which the coding region of the M gene including the R1 region and the R2 region of the M gene was completely deleted was prepared by PCR. The primers combined with the sequence of the upstream region and the downstream region of the M gene with the deletion of the M gene were synthesized, and multiplex PCR was performed to construct ΔM / rhPIV2 with the complete deletion of the coding region of the M gene. Among the primer sequences, the sequence containing a restriction enz...
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