Improved AS1411 nucleic acid aptamer and EVs coupled by using same
A technology of AS1411, 5T-AS1411, applied in the field of tumor biotherapy, can solve the problems of lack of targeting and limited application effect of EVs in extracellular vesicles
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[0043] The present application will be further explained below in conjunction with the accompanying drawings and embodiments. Before introducing the specific embodiments, the experimental background of some biological materials, experimental reagents, experimental equipment, etc. involved in the following embodiments is briefly introduced as follows.
[0044] biomaterials:
[0045] 4T1 cells, Shanghai Cell Bank, Chinese Academy of Sciences;
[0046] Balb / C mice (6-week-old, female, SPF grade) were purchased from Beijing Weitong Lihua Co., Ltd., and the feeding conditions were: 23-25°C, relative humidity 55%-60%, and the daily lighting time was maintained at 12 Hours (lights on at 8:00 am and off at 8:00 pm every day);
[0047] Cholesterol - 13 T bases - AS1411:
[0048]cholesterol-TTTTTTTTTTTT-TTGGTGGTGGTGGTTGTGGTGGTGGTGG),
[0049] Cholesterol - 9 T bases - AS1411:
[0050] cholesterol-TTTTTTTT-TTGGTGGTGGTGGTTGTGGTGGTGGTGG,
[0051] Cholesterol-9 T bases-AS1411 random s...
Embodiment 1
[0077] Before further introducing the specific technical solutions of the present application, in this example, the inventor first briefly introduces the extraction method of Escherichia coli outer membrane vesicles (OMVs) (Outer Membrane Vesicles) involved in the present application as follows.
[0078] The preparation of Escherichia coli outer membrane vesicle OMVs was specifically obtained by extraction as follows:
[0079] First, the preserved Enterobacter strains were inoculated on a solid LB plate containing 100 mg / mL ampicillin, and incubated upside down at 37°C for 12 h in a constant temperature incubator;
[0080] Subsequently, single clones were picked and transferred to 5 mL LB liquid medium containing 100 mg / mL ampicillin, cultured with shaking at 220 rpm at 37°C for 12 h; Penicillin 100 mL LB liquid medium, 37 ° C, 220 rpm shaking culture for 12 h;
[0081] Finally, take 50 mL of the bacterial liquid, centrifuge at 100,000 g for 30 min, discard the precipitate, a...
Embodiment 2
[0085] On the basis of the Escherichia coli OMVs prepared in Example 1, the inventors further coupled the cholesterol-modified nucleic acid aptamer AS1411 (the nucleic acid aptamer mainly has the targeting ability) to it, and in order to determine the Whether the base length between cholesterol and AS1411 affects the targeting ability of coupled OMVs, the inventors designed three base length AS1411 targeting systems: 5 T AS1411 (5T-AS1411), 9 T AS1411 (9T-AS141), 13 T AS1411 (13T-AS1411), and designed a 9 T random sequence AS1411 (9T-S-AS1411) as a control (random sequence AS1411 has no targeting ability), That is:
[0086] 5T-AS1411: cholesterol-TTTTT-TTGGTGGTGGTGGTTGTGGTGGTGGTGG,
[0087] 9T-AS141: cholesterol-TTTTTTTTTT-TTGGTGGTGGTGGTTGTGGTGGTGGTGG,
[0088] 9T-S-AS1411:cholesterol-TTTTTTTTTT-ATCGATCGATCGATCGATCGATCGATCGA,
[0089] 13T-AS1411: cholesterol-TTTTTTTTTTTTTT-TTGGTGGTGGTGGTTGTGGTGGTGGTGG),
[0090] When coupling the above-mentioned nucleic acid aptamers with ...
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