Modified mRNA sequence for increasing uric acid excretion and application thereof
A uric acid and sequence technology, applied in the field of molecular biomedicine, can solve the problems of hindering the reabsorption of proximal convoluted tubules, liver and kidney damage, etc., and achieve the effect of increasing the effect of uric acid excretion, reducing blood uric acid in the body, and having no adverse reactions.
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Embodiment 1
[0060] Example 1 Construction of the recombinant plasmid pET-28a(+)-ABCG2 carrying the ABCG2 DNA sequence The DNA sequence of mouse ABCG2 was found from NCBI, and the design added 5'UTR and 3'UTR, which contained T7 promoter, Kozak sequence, and Add BamHI and Sal I restriction sites, respectively. The sequence was synthesized by Shanghai Shenggong Company. The synthetic sequence and the pET-28a(+) plasmid were simultaneously digested with BamH I and Sal I. 50μL enzyme digestion system: BamHI 1μL (10U·μL), SalI 1μL (10U·μL), 10×K Buffer 5μL, DNA 20μL, ddH 2 O 23 μL. Constant temperature water bath 37 ℃, 2h. After purification, use T4 DNA ligase to ligate the digested pET-28a(+) plasmid with the target gene fragment, see figure 1 .
Embodiment 2
[0061] Example 2 Enzyme digestion identification of recombinant plasmid pET-28a(+)-ABCG2
[0062] The recombinant plasmid was transformed into Escherichia coli DH5α by heat shock method, spread evenly on the LB screening plate containing kanamycin, and cultured overnight at 37°C. A single colony was shaken to amplify and extract the recombinant plasmid for identification by double-enzyme digestion electrophoresis. The results showed that ABCG2 sequence with 5'UTR and 3'UTR was successfully inserted into pET-28a(+) plasmid. See figure 2 .
Embodiment 3
[0063] Example 3 In vitro transcription of ABCG2 mRNA and Western bolt detection of mRNA protein expression in TCMK-1 cells
[0064] Using the recombinant plasmid pET-28a(+)-ABCG2 as a template, the target gene sequence was amplified by PCR as template DNA for in vitro transcription. 120 Ts were added to the end of the downstream primer sequence for tailing. The PCR product was electrophoresed on 1.2% agarose gel, and the target gene was extracted and purified using a DNA gel extraction kit. The purified DNA samples were sent to Shanghai Sangon Company for sequencing and identification to ensure that the sequences were consistent and the tails were successfully added before in vitro transcription. According to the capping system in the MEGAscript T7 kit instruction manual, the DNA product with poly(T) tail added as a template was transcribed in vitro under the action of T7 RNA polymerase to obtain ABCG2 mRNA, and the mRNA was added with 3′-O-Me - m7G(5')ppp(5')G RNA cap anal...
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