1 typerecombinant adeno related viral vaccine of HPV16 and method of producing the same
A virus vaccine and virus technology, applied in the field of HPV16 type 1 recombinant adeno-associated virus vaccine and its preparation, can solve the problems of low expression level of L1 protein, and achieve the effect of increased titer and good preventive effect
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[0018] Experimental example 1: Construction of a recombinant adeno-associated virus containing the nucleic acid sequence shown in SEQ ID No:1
[0019] 1. Construction of a type I recombinant adeno-associated virus shuttle plasmid containing the nucleic acid sequence shown in SEQ ID No:1
[0020] (1) Preparation of E. coli DH5α competent cells:
[0021] Pick a single colony of DH5α from a fresh plate cultured at 37°C for 16-20 hours, inoculate it in 5ml of antibiotic-free LB medium, and culture it overnight (12-16h) at 37°C with vigorous shaking. Transfer 0.5ml from the above culture on the next day and transfer it to 50ml LB medium at 1:100 and continue to culture for about 3h. When the OD600 of the bacterial solution is 3, transfer the bacteria to a sterile In a 50ml centrifuge tube pre-cooled on ice, leave it in an ice bath for 30 minutes. Centrifuge at 4000 rpm for 10 min at 4° C., discard the supernatant, invert the tube for 1 min to drain the residual culture solution, and re...
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[0042] Experimental example 2: Identification of recombinant adeno-associated virus rAAV-mod.HPV16L1, virus titer analysis and electron microscope observation
[0043] (1) Expression of mod.HPV16L1 gene in recombinant adeno-associated virus:
[0044] Infect 1×10 with rAAV-mod.HPV16L1 virus of 10 MOIs 6 293 cells, add sodium butyrate with a final concentration of 10mmol / L at the same time, scrape the cells after 48h, wash twice with ice-cold PBS, extract the total cell protein according to the instructions of TRIzol reagent, and dissolve it in 1% SDS solution, 0.1% SDS was dialyzed 3 times, centrifuged at 10,000 g at 4°C for 10 min, and the supernatant was collected. Adjust the total protein content of BCA cells to 5mg / ml. The total cell protein extracted with 50μg TRIzol was subjected to SDS-PAGE electrophoresis and transferred to the membrane. The mouse anti-HPV 16L1 monoclonal antibody (camvir-1) was used as the primary antibody, and the horseradish enzyme-labeled goat anti-mous...
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[0049] Experimental example 3: Preliminary study on the immune effect of recombinant adeno-associated virus rAAV-mod.HPV16L1:
[0050] 1. Immunization of mice
[0051] Twenty female C57BL / 6 mice aged 4-6 weeks were randomly divided into 2 experimental groups, 5 mice in each group, namely rAAV-mod.HPV16L1 group; those containing the nucleic acid sequence shown in SEQ ID No:1 Recombinant adenovirus rAd-mod.HPV16L1. The corresponding rAAV-EGFP and rAd-EGFP control groups were set up in each experimental group according to the vaccination route (the control viruses were purchased from the original Zhengyang Gene Technology Co., Ltd.). The dosages used are rAAV 1×10 11 vg / only; rAd 1×10 7 IU / head, bilateral tibialis anterior muscle was injected, and each group was immunized once. All experimental operations were performed under anesthesia in mice. On the 12th week after immunization, serum and vaginal secretions of each group of mice were collected for use.
[0052] 2. Detection of neu...
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