Nanogene drug targeting interleukin-22 for treating liver injury
A technology of interleukin and gene medicine, applied in the field of biopharmaceuticals, to achieve the effect of reducing immunogenicity
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Embodiment 1
[0039] Example 1. Preparation of interleukin 22 and apolipoprotein AI recombinant fusion gene
[0040] Obtaining the gene encoding interleukin 22: human peripheral blood mononuclear cells were cultured in RPMI1640 medium containing 5 μg / ml lipopolysaccharide for 18 hours, RNA was extracted with Trizol, and RNA was reversed with a reverse transcription kit , finally obtain cDNA;
[0041] Use the cDNA obtained by reverse transcription as a template, and use Primer to design primers, where the upstream primer is: 5'-agtt CATATg gcgcccatcagctcccact-3',) where the underline is the NdeI restriction site; the downstream primer is: 5'-atcg GGATCC aatgcaggcatttctcagag-3', where the underline is the BamHI restriction site. Primers were synthesized by Shanghai Sangon Bioengineering Company;
[0042] The interleukin 22 gene containing NdeI and BamHI restriction sites was cloned by PCR method, and detected by 1% agarose gel electrophoresis. The size of the PCR product was about 500bp,...
Embodiment 2
[0046] Example 2, construction of interleukin 22 and apolipoprotein AI recombinant fusion gene expression vector
[0047] Take pVAX1 expression vector and pMD19-T-interleukin 22 encoding gene plasmid, use NdeI and BamHI restriction endonucleases to carry out double enzyme digestion, and use 1% agarose gel electrophoresis to separate the digested products, use gel recovery kit Recover the target fragment, among which the pVAX1 expression vector recovers a fragment of about 3000bp, and the pMD19-interleukin 22 recovers a fragment of about 500bp;
[0048] The ligation reaction is carried out under the catalysis of T4 DNA ligase. The molar ratio of pET24b expression vector restriction fragment and interleukin 22 gene target fragment is about 1:3. The ligation reaction system is 25ul, including T4 ligase 1ul, 10XT4 DNA ligase buffer 2.5ul solution, connect overnight at 16°C;
[0049] The ligation product was transformed into competent Escherichia coli Top10, spread on a kanamycin-...
Embodiment 3
[0053] Example 3pIA in vitro and in vivo expression experiments
[0054] HEK-293T cells were planted in a six-well plate, and when the cell fusion rate reached 70-80%, 2 and 4 μg of pIA were transfected with lipofectamine2000, respectively. The cells continued to be cultured for 24 hours. After the cells were harvested and lysed with a cell lysate, they were quantified with a BCA kit, and Western blot was used to detect the contents of interleukin 22 and ApoA I in the cells (such as figure 1 B, C shown);
[0055] HEK-293T cells were planted on sterilized coverslips. When the cell confluence rate reached 70%, 4 μg of pIA was transfected with lipofectamine 2000. After the cells were cultured overnight, the medium was discarded and replaced with 4% paraformaldehyde After fixing for 15 minutes, the cells were washed 3 times with PBS, then permeabilized with a solution containing 0.5% Triton X-100 for 20 minutes, and washed 3 times with PBS. Cells were then blocked with FBS for 2...
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