A Newcastle Disease Virus Recombinant Vaccine Strain Inserted into H7N9 Ha Protein
A technology of Newcastle disease virus and protein, applied in the direction of antisense single-stranded RNA virus, virus, vaccine, etc., can solve the problems of low antibody level, unsatisfactory immunogenicity, low expression efficiency of recombinant virus, etc., and achieve a good protective effect Effect
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Embodiment 1
[0021] The construction of embodiment 1 full-length cDNA cloning vector
[0022] According to the whole genome vector construction method established earlier, on the basis of the transformation of P gene and F gene, in the process of segment connection, fragment 2 was transformed by Over-lap PCR method, that is, in the NDV whole genome sequence 3171nt Then introduce the Pme I restriction site ( figure 1 ), and the constructed genome-wide vector was named PBRT-NDV-T. According to the sequence of the HA gene of H7N9 published by NCBI (SEQ ID NO: 1):
[0023]
[0024]
[0025] Codon optimization was carried out and sequences were synthesized, and the synthesized HA gene included codon-optimized and codon-unoptimized sequences. Among them, the H7N9 HA gene removes the 15bp base of the head and the 33bp base of the tail, retains the domains with better immunogenicity in the HA1 protein and the HA2 protein, and removes part of the signal peptide and transmembrane region sequ...
Embodiment 2
[0031] The rescue of embodiment 2 recombinant virus
[0032] When DF1-T7 cells were inoculated in a 35mm six-well plate and grown to 70%-80% monolayer, the transcription plasmids PBRT-NDV-2a, PBRT-NDV-2b and three auxiliary plasmids PCI-NP, PCI-P and The DF1-T7 cell line was co-transfected with PCI-L, using a calcium phosphate transfection kit, and the operation was performed according to the kit instructions. After 3-5 days of transfection, the culture supernatant was harvested and inoculated into the allantoic cavity of 9-11 day old SPF chicken embryos, cultured for 3-5 days, and the allantoic fluid of chicken embryos was taken to measure the HA titer. Harvest the allantoic fluid with positive HA test results, and freeze it at -70°C after aliquoting. The rescued recombinant NDVs containing the H7N9 HA gene were named rNDV-H7 a (codon-optimized, truncated) and rNDV-H7 b (codon-unoptimized, truncated).
[0033] Harvest the allantoic fluid 4 days after embryo inoculation with...
Embodiment 3
[0034] Growth characteristics of embodiment 3 recombinant virus in chicken embryo
[0035] The Newcastle disease virus rSL strain without HA gene and the recombinant Newcastle disease virus allantoic fluid containing HA gene were divided by 10 5 EID 50Inoculate the allantoic cavity of chicken embryos with SPF. The allantoic fluid was harvested at 24h, 48h, 72h and 96h after inoculation, and SPF chicken embryos were randomly collected at each detection time point, and the virus content per milliliter of allantoic fluid was detected (EID) 50 . Draw the growth curve, see the results image 3 As shown, the growth characteristics of the virus were consistent. It shows that after the HA gene is inserted, the growth rate of NDV is not affected, and the growth characteristic of high titer is still maintained.
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