Rabies virus vaccine and preparation method thereof
A technology of rabies virus vaccine and rabies virus, which is applied in the field of biomedicine, can solve the problems of limited virus carrier capacity, inability to replicate, and inconsistency, and achieve the effect of improving the ability to resist virus infection
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Embodiment 1
[0029] Embodiment 1: recombinant VSV-RABV G Vaccine construction and in vitro expression function identification
[0030] 1.1) Plasmid pXN2-G RABV Construction: The pXN2 vector contains the G gene sequence between the xbaI and MluI restriction sites, so we entrusted Jinweizhi to synthesize the XbaI-MluI fragment, in which the VSV G gene is replaced by the G gene of the RABV epidemic strain, and xbaI and MluI restriction sites were connected to the pXN2 vector to obtain pXN2-G RABV Plasmids, verified by sequencing, figure 1 is the build schematic.
[0031] 1.2) Recombinant VSV-RABV G Preparation and expression detection: BHK-21 cells were first infected with poxvirus with T7 RNA transcriptase, and 2 hours after infection, the cells were infected with pXN2-G RABV , pP, pL and pN plasmids were co-transfected in a ratio of 10:5:4:2. Three days after transfection, cytopathic changes were observed, and the supernatant of the cells was collected to infect Vero cells again to am...
Embodiment 2
[0032] Embodiment 2: recombinant VSV-RABV G Vaccine-Induced Specific Antibody Responses
[0033] 2.1) Mouse nasal drop immunization method and serum collection
[0034] The BALB / c male mice of 6-8 weeks were divided into 3 groups, and the 2 groups were immunized with nasal drops: 10 6 PFU VSV-GFP (negative control), 10 6 PFU VSV-RABV G , 1 group intramuscular injection of 10 6 PFU LBNSE (attenuated vaccine), 6 mice per group. The procedure of intranasal immunization was as follows: 80-120 μl of 0.75% pentobarbital sodium was intraperitoneally injected into the mice to slightly anesthetize the mice. The PBS virus vaccine solution containing VSV-GFP was instilled dropwise into the nasal cavity on both sides of the mouse, and VSV-RABV G Groups take the same approach. On the 10th day, 20th day and 30th day after immunization, blood was collected through the retro-orbital canthus vein, left at 37°C for 30min, centrifuged at 6000rpm for 10min, the serum was collected, subpack...
Embodiment 3
[0038] Example 3: In a mouse infection model, recombinant VSV-RABV G Vaccine Protection Response Detection
[0039] Divide 6-8 week old BALB / c male mice into 3 groups, 10 in each group, and the mouse immunization method is as described in 2.1.
[0040] At the 13th week after immunization, we infected mice with intracerebral injection, and observed the survival of mice within 20 days after infection. We found that the VSV-GFP immune group began to die 8 days after infection, and all the mice died by 13 days; while the final survival rate of the mice in the LBNSE group was 60% (6 / 10), VSV-RABV G Although the mice in the group were detected lower than the LBNSE group in the antibody level, they all survived in the challenge experiment (n=10), which may be due to the induction of more neutralizing antibodies ( Figure 4 ). Because the mouse challenge experiment was at 13 weeks after immunization, our results also suggest that VSV-RABV G Can provide a long-term protective immun...
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