Sealing TGF-betal antisensedingonucleotides and its use in preparing medicine for treating fibrillation related disease
An oligonucleotide and TGF-technology, applied in the field of new drugs to prevent fibrosis and treat fibrosis-related diseases, can solve the problems of poorly understood and difficult effects of cellular factors
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Embodiment 1
[0032] 1. TGF-β 1 Preparation of mRNA: RT-PCR amplification containing mature TGF-β 1 The cDNA fragment of the protein and signal peptide, the length of the cDNA is 1.3kb, its 5' end contains a Hind III restriction site, and its 3' end contains an XbaI restriction site. The correct sequence was verified by cloning into the A / T vector and sequencing. Carry out Hind III-Xba I double enzyme digestion to the vector containing the target fragment respectively, and recover the gene fragment; pSP64(polyA) vector (Promega) is subjected to Hind III-Xba I double enzyme digestion, and after recovery, it is ligated with the above gene fragment overnight to obtain TGF-β with SP6 promoter 1 cDNA(811-2170) in vitro expression plasmid pSP64-TGF-β 1 (see picture 1). The expression plasmid was constructed as figure 2 shown. Linearized pSP64-TGF-β digested with EcoR I 1 In vitro transcription was performed as a template, and the mRNA was recovered and stored at -70°C for later use.
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Embodiment 2
[0038] 1. TGF-β 1 Preparation of mRNA: same as Example 1.
[0039] 2. Design and synthesis of ssDNA random library: 2 ssDNA random libraries were designed and synthesized, with random sequences of 17 and 26 bases in length and wing lengths of 20 and 32 bases; synthesized by Espec Oligo Service Corp. in Japan, and the synthesis scale More than 6nmol, more than 98% of the full-length sequence.
[0040] Its specific composition is: 5'-accagcggactcgtgc-N 26 -cagacgactcgcccga-3'
[0041] 5'-accagcggac-N 17 -actcgcccga-3'
[0042] 3. Target TGF-β 1 Oligonucleotide screening of mRNA: same as Example 1.
[0043] 4. Cluster analysis of antisense oligonucleotide sequences: same as in Example 1.
[0044] 5. Optimization of antisense oligonucleotides: Through the screening of 3 random oligonucleotide libraries, it was found that the ssDNA library with a random sequence length of 20 bases and a wing length of 32 bases had the highest binding efficiency, and had the hig...
Embodiment 3
[0046] 1. Synthesis of thio-modified antisense oligonucleotide sequences: synthesized by an automatic DNA synthesizer and thio-modified during the synthesis process, the synthesis scale is 15OD, and purified by gel cutting after PAGE denaturing gel electrophoresis.
[0047] 2. Activity evaluation of antisense oligonucleotides: human oral epidermal carcinoma cells (KB) were seeded in 96-well plates, 4×10 3 Cells / well, 37°C, 5% CO 2 Incubate for 18-20h. After 60-80% cell confluence, use Opti-MEMI Reduced Serum Medium (Gibco) in serum-free state, liposome Oligofectamin (Invitrogen) for cell transfection, the total volume is 100μl, and the final concentration of antisense oligonucleotide is 200nmol / L. The above 18 sequences were transfected into KB cells respectively, and after 5 hours of action, 50 μl of Opti-MEM I Reduced Serum Medium containing 6% fetal bovine serum was added and cultured for 24 hours. The cells added with culture medium alone were used as blank control, th...
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