Novel application of nucleic acid vaccine expressing hepatitis B surface antigen
A surface antigen, hepatitis B virus technology, applied in the direction of antiviral agents, medical preparations containing active ingredients, antibody medical ingredients, etc., can solve problems such as insufficient immunogenicity and inability to induce cellular immunity.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0041] Detailed preparation method of nucleic acid vaccine and its gene sequence.
[0042] 1) Construction of HBV surface antigen nucleic acid vaccine ( figure 1 )
[0043] Based on the gene sequences of more than 20 HBV adr subtype surface antigens on Genbank, we designed the consensus sequence adr-HBs-Cons of HBV adr subtype surface antigen, and according to the sequence of higher mammals, especially humans (Homo sapiens) The codon preference was optimized with Accelrys software to enable better expression in eukaryotic cells without changing the primary structure of the adr-HBs-Cons protein. Our codon optimization also avoids modification of ciseopathic element sequences such as TATA boxes, ribosome entry sites, AT- or GC-rich sequence sites, sequence elements, potential splicing codons, etc. The gene sequence, shown as SEQ ID NO.1, was synthesized by Geneart Company of Germany (Regensburg, Germany), and loaded into vector pGA4 to obtain plasmid pGA4 / adr-HBs-cons / opt. Us...
Embodiment 2
[0045] Example 2. The target plasmid was extracted with Qiagen Plasmid Mega Kit produced by Qiagen.
[0046] 1) Aspirate 5 μL of the correctly identified bacterial preservation solution and inoculate it into 5 ml of LB culture solution containing kanatoxin, grow overnight at 37° C., 200 rpm. The next day, move the bacteria to a 250ml centrifuge bottle, centrifuge at 6000g at 4°C for 15min, discard the supernatant, and collect the bacteria.
[0047] 4) Resuspend the bacterial pellet in 50ml of buffer P1 and shake repeatedly until no bacterial clumps can be seen and all the bacteria are resuspended in the solution.
[0048] 5) Add 50ml of buffer P2 into the centrifuge bottle, gently invert it 5-6 times, the solution turns blue, and let it stand for 5 minutes.
[0049] 6) Add 50ml of buffer P3 into the above mixed liquid, gently invert 5-6 times, the blue solution disappears, the solution is layered, the upper layer is a solid milky white mass, and the lower layer is a clear liq...
Embodiment 3
[0059] Example 3. Cell Transfection
[0060] 293T cells were cultured in double-antibody DMEM high-glucose medium containing 10% fetal bovine serum at 37°C, 5% CO 2 Cultivate in a saturated humidity incubator until the logarithmic growth phase, digest with 2.5g / L trypsin, and use 5.0×10 6 Cells (8 mL) were inoculated in 100 mm culture dishes, and transfection began when they grew to 80% confluence. Cell transfection was carried out according to the PEI transfection method. Take 75 μL of PEI and add it to 750 μL of double-antibody-containing DMEM high-glucose medium, and another 15.0 μg of pSW3891 / adr-HBs-cons / opt recombinant expression plasmid was added to the above culture medium. Gently mix, incubate at room temperature for 15 minutes, then add the PEI / DNA complex to the culture flask and shake gently to mix evenly. At the same time, cells transfected with pSW3891 empty plasmid are used as a negative control, and can be replaced after 10 hours without serum containing doubl...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More