A kind of human gamma-interferon virus vector and its application
A viral vector and interferon technology, applied in the field of biomedicine, can solve the problem of weak antiviral effect of γ-interferon adenovirus, and achieve the effects of strong antiviral activity and antitumor activity, high biological activity, and simplified design
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Embodiment 1
[0036] Example 1 Human gamma-interferon virus vector
[0037] The signal peptide sequence and the selected human interferon gamma truncated sequence were optimized for codon bias, 5' region, DNA repeat sequence, mRNA secondary structure, GC content, SD sequence and other parameters to obtain the fusion gene Nucleotide sequence, and synthesized gene, the optimized nucleotide sequence of human gamma-interferon fusion gene is shown in SEQ ID NO:3.
[0038] The optimized human γ-interferon fusion gene and PDC316 plasmid (Microbixbiosystem) were used to construct a recombinant plasmid. Through PCR amplification, gel recovery, enzyme digestion, connection, transformation, PCR, enzyme digestion and sequencing identification after extracting the plasmid, the sequenced correct plasmid is the recombinant human gamma-interferon shuttle vector.
[0039] The recombinant human γ-interferon shuttle vector plasmid and adenovirus packaging helper plasmid (PBHG) were co-transfected into HEK293A ...
experiment example 1
[0042] Experimental example 1 Determination of human gamma-interferon expression activity
[0043] Plate a 6-well plate at a density of 80,000 cells per well, store at 37°C, 5% CO 2 After culturing in the incubator for 20 h, discard the culture medium. WISH cells were transfected with recombinant adenoviruses with MOI values of 100 (Example 1 and Comparative Example 1) and 100 MOI Ad / LacZ (as an adenovirus empty vector control), the virus was diluted with serum-free DMEM culture medium, and three replicates were performed. Well, 37°C, 5% CO 2 Conditioned for 2h. The virus liquid was discarded, and DMEM medium containing 5% newborn bovine serum was added to each well to continue culturing. Pipette the cell culture supernatant from each well into a centrifuge tube, and centrifuge at 4000rpm for 10min. One of the test samples in each well was diluted 200 times and then detected by the human interferon-γ detection kit. The test results are shown in Table 1 below.
[0044] ...
experiment example 2
[0047] Experimental Example 2 Determination of biological activity of human gamma-interferon
[0048] The biological activity of the human γ-interferon virus prepared in the present invention was detected according to the general rule 3523 of the 2020 edition of the "Chinese Pharmacopoeia": interferon biological activity assay.
[0049] The specific detection steps are as follows:
[0050] Preparation of the standard solution: take the national standard for the determination of the biological activity of human interferon, redissolve it according to the instructions, and dilute it with the measurement culture medium to contain 1000IU per 1mL. In a 96-well cell culture plate, make 4-fold serial dilutions, a total of 8 dilutions, and make 2 wells for each dilution. Operate under sterile conditions.
[0051] Preparation of the test solution: after dissolving the test product according to the marked amount, dilute it with the measurement culture medium to contain about 1000IU per...
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