Construction of F gene replaced chimeric measles attenuated strain
A technology of attenuated strains and measles virus, applied in genetic engineering, inactivation/attenuation, plant genetic improvement, etc., can solve problems such as insufficient antibody titer, measles human health threat, and rapid antibody attenuation
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Embodiment 1
[0135] Example 1: Construction and identification of chimeric virus full-length cDNA
[0136] Such as figure 1 Shown is the chimeric measles virus skeleton.
[0137] With the cDNA reverse-transcribed of the MV-1 strain measles virus nucleic acid as a template, use specific primers MV-F-F and MV-F-R in Table 1 to amplify its F gene fragment (comprising the F gene 5'UTR and the F gene 3'UTR), using the S191 full-length plasmid as a template, using specific primers TY-HN-F and FH-ZL-R to amplify its F gene deletion vector by PCR, and then obtain the target gene by one-step homologous recombination plasmid.
[0138] It was confirmed by enzyme digestion that the full-length cDNA of the chimeric virus was successfully cloned, and the results were as follows figure 2 shown.
[0139] Table 1 The specific detection primers of chimeric virus
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Embodiment 2
[0142] Example 2: Chimeric virus rescue
[0143] Experimental method and steps:
[0144] Cells were seeded in 6-well plates, and when the cells grew to about 80%, full-length plasmids PAC-S-191, PAC-S-191-F and related auxiliary plasmids pcDNA3.1-N, pcDNA3.1-P, 293T cells were co-transfected with pcDNA3.1-L and pCAGGS-T7. After 6 hours, the medium was discarded and the medium containing 2% FBS fetal bovine serum was added. After 4 days of culture, the cells were passaged at a ratio of 1:3 and an equal amount of Vero-SLAM cells were added until the cells became confluent to collect the virus supernatant.
[0145] A large number of pMV full-length clones and helper plasmids were extracted; they were co-transfected into cells for virus rescue.
[0146] 293T cells were inoculated in a six-well plate for overnight culture, and cell wells with a cell confluence of 80-90% were selected for virus rescue.
[0147] The specific process is as follows: the full-length plasmid pMV or pM...
Embodiment 3
[0152] Example 3: Analysis of in vitro properties of chimeric viruses
[0153] In order to evaluate the growth characteristics of the chimeric virus in vitro, in this example, the multi-step growth curves of the chimeric virus and its parent virus were studied in Vero and Vero-Slam cell lines, respectively.
[0154] First, make a cell suspension with a cell concentration of 1.0×10 5 -5.0×10 5 Between cells, the cells are divided into 2×10 5 cells / well were seeded in a 12-well plate.
[0155] The next day, when the cells are about 90% full, wash the cells with PBS, inoculate 1ml of virus at 0.01 MOI, incubate at 37°C for 1 hour, discard the virus solution, rinse twice with PBS, and replace the maintenance solution. Sampling was then carried out every 24 hours for 5 consecutive days. Virus titers were then detected by the microcytopathic assay.
[0156] The result is as Figure 5 As shown, in Vero cells, the titer of the chimeric virus rMV / F(H1a) was slightly higher than t...
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