Anti-osteoarthritis recombinant miR-140 and production method and application thereof
A production method and arthritis technology, applied in the fields of molecular biology and medicine, can solve the problems of limited development of functional RNA therapy and high cost of RNA raw materials, and achieve the effect of convenient equipment, low price, and maintenance of anabolic balance
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Embodiment 1
[0034] Example 1: The recombinant miR-140 expression plasmid was constructed with the pBSKrnaSeph / has-mir-34a vector, and the recombinant miR-140 was expressed.
[0035] (1) Design primers according to the effective sequence of recombinant miR-140 (SEQ ID NO: 1) and the sequence on the pBSKrnaSeph / has-mir-34a expression vector, and add 1-15nt vector insertion sites at both ends of the primers Homologous sequences on both sides.
[0036] Table 1 Recombinant miR-140 primers
[0037]
[0038] (2) Synthesis of insert fragments
[0039] Using the two primers in Table 1 as templates, insert the precursor sequence of recombinant miR-140 into the pBSMrnaSeph plasmid by using the enzyme cutting site at the anticodon loop of the tRNA (SEQ ID NO: 3) of the pBSMrnaSeph plasmid, Construct the expression vector; the reaction system is shown in Table 2, and the reaction process is shown in Table 3:
[0040] Table 2 Polymerase in vitro amplification chain reaction system (50 μL)
[0041]...
Embodiment 2
[0059] Example 2: Expression of recombinant miR-140
[0060] (1) After 200 ng of recombinant miR-140 expression plasmid was transformed into HST08 competent bacteria, 5 mL of LB medium was added, and cultured overnight at 37° C. with shaking at 200 rpm. After the bacterial solution was centrifuged at 10000 g for 2 min, the precipitate was collected. Add 180 μL of 10 mM magnesium acetate-Tris·HCl solution to the precipitate to resuspend, then add 200 μL of saturated phenol, and shake at room temperature for 20-60 min. After centrifuging at 10000 g for 10 min, the aqueous phase was collected, and 5M NaCl of 0.1 times the volume of the aqueous phase was added to precipitate macromolecular impurities. Add 2 times the volume of absolute ethanol to the supernatant, centrifuge at 10000 g for 10 min, and discard the supernatant. Blot the residual ethanol with absorbent paper, add DEPC water to dissolve the RNA after the RNA is dry, measure the concentration, and store in a -80°C ref...
Embodiment 3
[0064] Embodiment 3: FPLC purifies recombinant miR-140
[0065] (1) Using Bio-Rad NGC TM Chromatography System, with ion exchange column (ENrich TM Q10×100Column) to purify recombinant miR-140.
[0066] Mobile Phase A: 10mM NaH 2 PO 4 Solution, pH 7.0. Mobile phase B: 10mM NaH 2 PO 4 Solution, 1M NaCl solution, pH7.0.
[0067] The flow rate was 2.0 mL / min. Wash the chromatographic column alternately with DEPC water, mobile phase A, and mobile phase B for about 1 h. 5 column volumes per flush.
[0068] Run the following program to separate total RNA: 0~8.9min (0%B), 8.9~13.7min (55%B), 13.7~53.7min (55~75%B), 53.7~73.7min (75~85%B) B), 73.7-83.7min (100%B), 83.7-93.7min (0%B). RNA was detected by absorbance at 260 nm, and peaks corresponding to recombinant RNA were collected. The purity was confirmed by denaturing polyacrylamide gel electrophoresis.
[0069] (2) RNA sample processing method
[0070] The total RNA extraction steps were the same as above. After th...
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