Compound type therapeutic hepatitis B gene vaccine and construction method thereof
A technology of genetic vaccine and construction method, applied in the field of hepatitis B vaccine, can solve the problem of not being able to completely remove intracellular infection virus, etc., and achieve the effect of good intracellular virus and removal of intracellular virus
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[0025] 1. PCR primer design and synthesis
[0026] Two pairs of primers were designed as the primer sequences for amplifying T-bet and HBV-L respectively. See Table 1 for the primer sequences and restriction sites. The sizes of the amplified T-bet and HBV-L gene fragments were 1608bp and 1203bp, respectively. Primers were synthesized by Invitrogen Company.
[0027] Target gene amplification primer sequence
[0028]
[0029] 2. PCR amplification of the target gene
[0030] 2.1 RT-PCR method to clone human T-bet gene
[0031] 2.1.1 Extraction of total RNA from human peripheral blood lymphocytes
[0032] (1) Take 5ml of anticoagulated blood from a healthy person with a blood collection tube, centrifuge at 3000rpm for 5min, and suck out the serum.
[0033] (2) Take a 15ml centrifuge tube and add 2ml Ficoll.
[0034] (3) Add 1ml PBS to the blood collection tube to resuspend the cells, attach to the wall at 45 degrees and add to a centrifuge tube equipped with Ficoll, and c...
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