shRNA (short hairpin ribonucleic acid) capable of inhibiting IRS (insulin receptor substrate) 2 gene expression and application thereof
A gene expression and product technology, applied in the field of genetic engineering, can solve the problems that can not be used as long-term disease research targets, mice have short lifespan, and mice cannot simulate human diseases well
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Embodiment 1
[0022] Cloning and screening of porcine IRS2 gene
[0023] (1) Cloning of the 3'UTR region of the pig IRS2 gene
[0024] The human IRS2 gene sequence is known. Compare the human IRS2 gene with the pig genome, design primers according to the conserved region sequence, and use 2d Bama pig liver tissue cDNA as a template to clone the conserved sequence of the pig IRS2 gene. PCR Reaction system 20ul: cDNA 0.5ul, primer 2ul, rTaq 0.5ul, 10×PCR Buffer 2.5ul, dNTPs 2ul, dH 2 O17.5ul. The PCR reaction program was: 94°C for 5min; 35 cycles of 94°C for 30s, 59.3°C for 30s, and 72°C for 30s; 72°C for 10min. The results of PCR products were observed in 1% agarose gel electrophoresis, and the target band was recovered from the gel, connected to the PMD-18T cloning vector for sequencing. The mutation-free sequence was obtained for 3' RACE amplification, and the standard operation was performed according to the instructions of the Takara kit.
[0025] The results showed that the obtained...
Embodiment 2
[0027] Construction of p-Genesil-shIRS2 interference vector
[0028] According to the porcine IRS2 sequence obtained by cloning, four pairs of interference fragments were designed through the Ambion website (Table 1), and single-stranded shRNA interference fragments with BamHI and HindIII restriction sites were synthesized, and after renaturation into double strands, they were connected to Ⅰ and HindⅢ linearized p-Genesil1.0 vector. After the sequencing was correct, the successfully constructed vector was p-Genensil-shIRS2-1-4. The effective interference vector screened out is p-Genensil-shIRS2-1.
[0029] Table 1 Interfering fragment sequence of pig IRS2 gene
[0030]
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